calcium sparks

Calcium Sparks

HEPING CHENG AND W. J. LEDERER

Institute of Molecular Medicine, National Laboratory of Biomembrane and Membrane Biotechnology, Peking

University, Beijing, China; and Medical Biotechnology Center, University of Maryland Biotechnology Institute,

Baltimore, Maryland

I. Introduction 1492II. Microscopic Ca2� Gradients in Living Cells 1493

III. Ca2� Sparks 1493A. Spontaneous sparks 1495B. Evoked sparks 1497C. Sparks and EC coupling 1498D. Sparks and Ca2� waves 1498E. Sparks in different types of cardiac cells 1499F. Sparks in other types of cells 1500G. Terminology 1500

IV. Theory of Spark Formation and Detection 1500A. Spark formation 1500B. Spark detection 1500C. Ca2� spikes 1501D. Inferring [Ca2�]i gradients from sparks 1502

V. Spark Morphology 1502A. Amplitude 1502B. Width 1503C. Kinetics 1503D. Autonomy 1503

VI. Ca2� Sparklets 1504VII. Intermolecular Signaling Between LCCs and RYRs 1505

A. Triggering sparks by single LCC openings 1505B. Power law of spark activation 1506C. Kinetics of sparklet-spark coupling 1506

VIII. Ca2� Blinks 1507A. In cardiac myocytes 1507B. In skeletal muscle cells 1508

IX. Spark Mechanisms: Activation 1509A. Spontaneous sparks 1509B. Triggered sparks 1511

X. Spark Mechanisms: Coordination in a CRU 1511A. Overview 1511B. Key observations 1513C. CICR and coupled gating 1513D. How many RyRs open during a spark? 1514

XI. Spark Mechanisms: Termination 1514A. Local Ca2� depletion 1514B. Stochastic attrition 1514C. Desensitization of CICR by Ca2� store depletion 1515D. Coupled gating as a termination mechanism 1515E. Channel inactivation and adaptation 1515F. Refractoriness of local CICR 1516G. RyR gating in vivo versus in vitro 1517

XII. Ca2� Sparks in Heart Disease 1517A. Arrhythmic disease 1517B. Heart failure 1518C. Remodeling of the Ca2� signaling system 1518D. Genetic and acquired channel dysfunction 1520E. Cardiac Ca2� signaling in diverse diseases 1521

Physiol Rev 88: 1491–1545, 2008;doi:10.1152/physrev.00030.2007.

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XIII. Sparks and Embers in Skeletal Muscles 1521A. Overview 1521B. Sparks in amphibians 1521C. Embers and sparks in mammals 1523

XIV. Smooth Muscle Sparks 1524A. Activation of Ca2�-sensitive channels by subsurface sparks 1524B. Ca2� sparks relax smooth muscle 1525C. Spark modulation of membrane excitability 1526

XV. Neural Ca2� Sparks 1527A. Characteristics and mechanisms 1527B. Properties of CICR in DRG neurons 1527C. Possible role of neuronal sparks 1527

XVI. IP3R Ca2� Puffs and Blips 1529XVII. Sparkless Release 1530XVIII. New Insights Into Ca2� Signaling 1530

A. Mechanisms underlying excitation-Ca2� release coupling 1530B. Ca2� sparks and the CICR paradox 1531C. Cellular architecture of Ca2� signaling 1532D. The digital-analog dichotomy 1533E. Biochemistry of Ca2� sparks 1533F. Building complexity from simplicity 1534

XIX. Perspective 1534

Cheng H, Lederer WJ. Calcium Sparks. Physiol Rev 88: 1491–1545, 2008; doi:10.1152/physrev.00030.2007.—Thecalcium ion (Ca2�) is the simplest and most versatile intracellular messenger known. The discovery of Ca2� sparksand a related family of elementary Ca2� signaling events has revealed fundamental principles of the Ca2� signalingsystem. A newly appreciated “digital” subsystem consisting of brief, high Ca2� concentration over short distances(nanometers to microns) comingles with an “analog” global Ca2� signaling subsystem. Over the past 15 years, muchhas been learned about the theoretical and practical aspects of spark formation and detection. The quest for thespark mechanisms [the activation, coordination, and termination of Ca2� release units (CRUs)] has met unexpectedchallenges, however, and raised vexing questions about CRU operation in situ. Ample evidence shows that Ca2�

sparks catalyze many high-threshold Ca2� processes involved in cardiac and skeletal muscle excitation-contractioncoupling, vascular tone regulation, membrane excitability, and neuronal secretion. Investigation of Ca2� sparks indiseases has also begun to provide novel insights into hypertension, cardiac arrhythmias, heart failure, and musculardystrophy. An emerging view is that spatially and temporally patterned activation of the digital subsystem conferson intracellular Ca2� signaling an exquisite architecture in space, time, and intensity, which underpins signalingefficiency, stability, specificity, and diversity. These recent advances in “sparkology” thus promise to unify thesimplicity and complexity of Ca2� signaling in biology.

Ja, Kalzium das ist alles. . .Otto Loewi (1936 Nobel Laureate)

I. INTRODUCTION

Calcium signaling is paradoxically both simple andcomplex in that single-atom calcium ions (Ca2�) act asthe most versatile biological messenger known. Rapid,transient changes in Ca2� concentration directly controlmuscle contraction, cell locomotion, hormonal secretion,and neural transmission. Sustained elevations of Ca2�

signals play pivotal roles in biological processes rangingfrom fertilization to gene expression to apoptosis (25–27,79, 309). Yet, being a single-atom cation, Ca2� exists inonly one biologically relevant form that undergoes neithercatabolic degradation nor anabolic synthesis. Its biologi-cal information-coding ability derives almost entirelyfrom its binding and unbinding from target proteins aswell as the electrical currents it generates when moving

across a biological membrane. This simplicity of Ca2�

signaling seems to be at odds with the complexity andexquisiteness of the processes that it controls. One ionconnecting thousands of proteins, each harboring one ormore evolutionarily conserved Ca2�-binding motifs (S.Wei, personal communication), has given rise to theenigma of how Ca2� can choreograph these molecularplayers to fulfill the amazingly diverse and even opposingphysiological functions in a given cell. As Ca2� is the bestcharacterized among common biological messengers[cAMP, inositol 1,4,5-trisphosphate (IP3), and nitric oxide(NO), for example], resolving the paradox of Ca2� signal-ing should be enlightening with respect to fundamentalprinciples of intracellular signal transduction.

Over the past two decades, vibrant research on localCa2� signaling in muscles, neurons, and other excitableand nonexcitable cells has laid the foundation for thisreview. Multiple studies by the authors and by many

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others have led to a body of work that addresses olddilemmas and raises new questions. In particular, thediscovery of discrete, elemental Ca2� signaling eventsexemplified by “Ca2� sparks” (Table 1) (69) has illumi-nated the building principles of the Ca2� signaling systemand brought teleological insights into many supramolecu-lar Ca2� signaling nanomachines in diverse types of cells.This work has started to delineate in some detail thecharacter of intracellular Ca2� signals in the dimensionsof space, time, and magnitude and has begun to unify thesimplicity of elementary Ca2� signaling (in terms of de-sign principles) and the versatility of physiological func-tions.

Here the authors seek to provide a critical review ofthe rapidly evolving field of “sparkology.” We recognize,however, that space limitations and the need for claritydemand that a tight focus be maintained. While we inves-tigate the findings and issues in the heart in great detail,we also present major observations and issues in skeletaland smooth muscles and oocytes and provide an overviewof recent findings in neurons, as well as other excitableand nonexcitable cells. It is our hope that the review willbe informative and provocative to both students and ac-tive investigators who are broadly interested in signaling.Table 1 is a collection of the names used for elementalCa2� signals and related structural components. See Ref-erences 15, 24, 38, 41, 68, 71, 79, 90, 140, 148, 173, 182, 200,245, 277, 278, 280, 309, 321, 334, 354, 364, 389, 403, 407 forrecent reviews on topics related to local Ca2� signaling.

II. MICROSCOPIC Ca2� GRADIENTS IN

LIVING CELLS

Sustained, macroscopic gradients of free Ca2� con-centration ([Ca2�]) occur across cell surface and intracel-lular membranes of organelles. A 104-fold [Ca2�] gradientexists across the plasma membrane that partitions theextracellular space from the cytosol. A similar gradient isfound between the lumen of the endoplasmic and sarco-plasmic reticulum (ER/SR) and the cytosol. The cytosolic[Ca2�] ([Ca2�]i) is actively maintained at a very low levelaround 100 nM by Ca2� homeostatic mechanisms, includ-ing the plasmalemmal Na�/Ca2� exchanger (NCX) andCa2�-ATPase, the ER/SR Ca2�-ATPase (SERCA), and by abattery of Ca2� buffering molecules of differing capacityand kinetics. A low resting cytosolic [Ca2�], while avoid-ing the toxicity of sustained high [Ca2�], bestows on theCa2� signaling system a wide dynamic range and a highsignal-to-background ratio.

Many Ca2� signaling cascades are initiated by rapidmobilization of Ca2� among membrane-bound compart-ments, usually down its electrochemical gradient acrossthe membrane. Ca2� entry from the exterior of the cell ismediated by diverse Ca2�-permeant channels and their

gating mechanisms, which include voltage, ligands, me-chanical force, temperature change, pH, reactive oxygenspecies (ROS), and intracellular signals arising from Gprotein coupling receptor (GPCR) activation, and evendepletion of the ER/SR store. NCX operating in the re-verse mode, i.e., translocating Ca2� inward and Na� out-ward, also brings Ca2� in under certain circumstances. Incontrast, Ca2� release from the ER/SR is mediated by onlytwo families of Ca2� channels, the ryanodine receptor(RyR) (121, 128, 255) and the IP3 receptor (IP3R) (21, 125),each with three major isoforms (types 1, 2, and 3). Re-cently, it has been suggested that presenilins form Ca2�

channels of tiny conductance, accounting in part for theconstant ER Ca2� leak (382). Restoration of Ca2� ho-meostasis following a transient change in [Ca2�] isachieved by extrusion across the plasma membrane byNCX and Ca2�-ATPase, and resequestration into theER/SR by SERCA, at the expense of biochemical andelectrochemical energy.

At the molecular level, transmembrane Ca2� translo-cation should be considered a discrete, stochastic pro-cess. When in action, Ca2�-permeant channels and trans-porters act as Ca2� sources (in the destination compart-ment) and sinks (in the source compartment), creatingdynamic [Ca2�] gradients in their immediate vicinity.Many determinants are thought to shape transient, micro-scopic [Ca2�] gradients (179, 186, 301, 344, 362). Theseinclude 1) Ca2� fluxes, their magnitudes, and time courses;2) intracellular Ca2� buffers, many of which are also Ca2�

effectors, their abundance, affinity, and kinetics;3) diffusion of Ca2� in the cytosolic milieu; and 4) spatialrestriction (212, 350). [Ca2�] gradients are accentuatedand sustained by spatial confinement, e.g., the subspaceof a couplon in muscles (Table 1), the tortuous ER/SRnetwork, synaptic terminals, dendritic spines, fine pro-cesses in neurons, and lamellipodia and filopodia in mi-grating cells. When the spatial scale of interest is compa-rable to the Debye length (�1 nm) of the negativelycharged lipid biomembrane, the electrostatic effect onCa2� distribution and diffusion also becomes evident(350). As we illustrate later, such nanodomain (1–100 nm)and microdomain (0.1–10 �m) Ca2� signals are of pro-found significance in determining signaling efficiency,specificity, and diversity.

III. Ca2� SPARKS

Ca2� signals in living cells were first “seen” in the late1960s as intracellular [Ca2�]i transients during singletwitches in barnacle muscle fibers (7, 157) and first im-aged in the late 1970s as Ca2� waves in fertilizing Medakafish eggs (136, 305). In these early experiments, [Ca2�]was measured with aequorin, the chemiluminescent indi-cator obtained from jellyfish (34). Shortly after, [Ca2�]i

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TABLE 1. Sparkology

Term Definition Comment

Ca2� releaseunit (CRU)

A CRU refers to a cluster of Ca2� release channels,either RyRs or IP3Rs or a mixture, in the ER/SRmembrane.

See Ref. 130. Functionally, a CRU includes the Ca2� release channels,the ER/SR cistern on which the release channel resides, andmolecular partners of the channel complexes on either side of orwithin the membrane.

Couplon Structural complex and functional unit of muscleEC coupling. Anatomic elements include thesarcolemmal Ca2� channels or voltage sensors,the CRU, and the subspace between them.Amphibian skeletal muscle couplon also containsparajunctional RyR3 arrays.

Redefined from Stern et al. (366), the original definition refers to theCRU only. Interchangeably used with “dyad” when referring to theanatomy. A triad contains two couplons. See also “Ca2� synapse.”

Subspace In a couplon, the space between the plasmamembrane and the ER/SR membrane at a dyad.In a Ca2� synapse, the space between apposingmembranes.

The subspace is the immediate, somewhat restricted space intowhich the Ca2� influx or the Ca2� release flux is delivered to itslocal targets, before it enters the bulk of the cytosol.

Subsurfacecistern(SSC)

Specialized region of the ER that comes into directapposition to the plasma membrane in a neuron,forming a structural feature reminiscent ofcouplons in muscles.

See Refs. 162, 311. EM picture of release channels on SSC yet to beseen.

Ca2� synapse Structural complex and functional unit thatmediates Ca2� signaling across apposedmembrane compartments.

See Ref. 363. Synonym to “couplon” in striated muscles. Generalizedto include the coupling sites between specialized SSC and theplasma membrane in other types of cells, and those between ERand mitochondria.

Ca2� spark An event and its fluorescent recording of Ca2�

release from a single CRU. Multiple meaningsmay be associated with this term in a context-sensitive manner: 1) the underlying local Ca2�

release flux, 2) the resultant local �Ca2��i

transient; in the presence of a Ca2� indicator, 3)the fluorescence spark in a cell (in object space),and 4) the observed fluorescence spark (inimage space).

See Ref. 69. Originally a name for RyR2-mediated events in cardiaccells, it is now used as a generic term for local Ca2� release eventsregardless of their molecular origin or cell type.

Ca2� syntilla Ca2� spark in a neuronal presynaptic terminal. See Ref. 95.Ca2� sparklet Local �Ca2��i transient that arises from a single

Ca2�-permeant channel.See Ref. 390. Originally defined for LCC.

Ca2� ember Long-duration, low-amplitude Ca2� release event. See Ref. 142. Synonym to RyR Ca2� sparklet when only a single RyRis involved. Its spatial spread is usually narrower than a spark.Lone embers are evoked by depolarization in mammalian skeletalmuscle cells (91). Trailing embers (ember in the decline phase of aspark) are induced by spark modifiers, such as ryanodine, FK506,rapamycin, Imperatoxin A, and bastadin 10, all of which producelong openings of the underlying release channels. See Refs. 69, 90,200, 420.

Ghost spark Spark appearing in treated adult skeletal musclewhich normally lacks spark activity.

See Ref. 394.

Ca2� glow Long-duration Ca2� spark. See Ref. 427. Reported in cultured superior cervical ganglion neuronsSynonym to “Ca2� ember” in Ref 171.

Ca2� burst Long-duration, compound Ca2� spark. See Ref. 401.Ca2� quark Ca2� release through a single RyR. See Refs. 230, 232. Synonym to RyR Ca2� sparklet.Ca2� quantum The signal unit that confers the regular peaks on

the histogram of Ispark.See Ref. 392. A quantal unit is thought to reflect a single RyR or a

tightly coupled subgroup of RyRs in a CRU.Compound

Ca2� sparkLocal release event that involves near-synchronous

activation of multiple adjacent CRUs.See Refs. 81, 415. Also called a “macrospark” (67).

Ca2� wave Regenerative Ca2� release that travels across manyCRUs. It usually reflects a high sensitivity ofCICR.

An aborting Ca2� wave or wavelet may appear as a cascade of Ca2�

sparks and compound Ca2� sparks in space and time. Spheric,planar, and spiral Ca2� waves can be seen in the same cell. SeeRefs. 62, 67.

Ca2� wavelet Abortive Ca2� wave. See Ref. 368.Ca2� spike Local or global �Ca2��i transient measured in the

presence of excess Ca2� buffers (e.g., EGTA).See Refs. 324, 357. Spike measurement can better resolve the location

and kinetics of Ca2� flux. Not to be confused with those referringto initial Ca2� surge during photolysis of caged Ca2� compounds(450) or sharply peaked whole cell Ca2� oscillations in neurons (22).

Ca2� puff Discrete local Ca2� release event of IP3R origin. See Ref. 428. Synonym to an IP3R Ca2� spark when a single CRU isinvolved or a compound IP3R Ca2� spark when multiple CRUs areinvolved. EM evidence for IP3R clusters or CRUs is still lacking.

Ca2� blip A very small Ca2� puff. See Ref. 369. A Ca2� puff or blip can be graded by varying IP3

concentration. A blip is thought to involve only one or a few IP3Rs.



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transients during cardiac excitation-contraction (EC)coupling were seen as aequorin signals in frog cardiacmuscle (2) and in canine Purkinje fibers (409). Measure-ment of [Ca2�]i transients began to seriously expand withmetallochromic dyes: first arsenazo III, used by Brownet al. (49) in squid axon and then in muscle, and laterantipyrylazo III used by Kovacs et al. (204). The creationby Tsien and co-workers of novel fluorescence indicatorslike fura 2 (147) catalyzed an explosion of new measure-ments and [Ca2�]i imaging activity in isolated smoothmuscle cells (413) and in single cardiac muscle cellsunder normal conditions (56) and during Ca2� overloadwhen propagating waves of elevated [Ca2�]i were ob-served (20, 410). Elegantly spiraling Ca2� waves were alsodocumented in Xenopus oocytes (217). The convergenceof two technical developments, the advent of a flexibleconfocal microscope (the BioRad MRC500), and the in-troduction of fluorescein and rhodamine-based Ca2� in-dicators (261) prepared the field for the discovery of eversubtler dynamic Ca2� signaling events. With the use offluo 3 as the Ca2� indicator (Table 2) in conjunction withconfocal microscopy, Ca2� sparks were first visualized inquiescent cardiac myocytes (Figs. 1 and 2).

A. Spontaneous Sparks

In unstimulated single cardiac myocytes, a Ca2�

spark appears abruptly amidst a seemingly featurelessbackground, reaches its peak of about a twofold increasein fluorescence intensity within 10 ms, and dissipates inanother 20 ms. It is confined to an area of �2.0 �m indiameter or �8 fl by volume. In a high concentration ofexogenous Ca2� buffer EGTA (4 mM), the local Ca2�

event corresponding to a spark is called a “Ca2� spike”(Fig. 3) which spans �0.6 �m, lasts �8 ms, and maybetter resolve the location and kinetics of the Ca2� fluxunderlying the spark (391). The characteristics of Ca2�

spikes are consistent with the idea that a Ca2� sparkarises from a “point” or narrow Ca2� source, a single Ca2�

release unit (CRU) (Table 1).The occurrence of spontaneous Ca2� sparks does not

require Ca2� entry into the myocyte through L-type Ca2�

channels (LCCs; also known as dihydropyridine recep-tors, DHPRs) or by other Ca2� paths across the plasmamembrane. Spontaneous Ca2� sparks at the resting po-tential (�80 mV) continue to be seen when extracellularCa2� is removed for a brief time (28, 58, 69). Similarly,

TABLE 1.—Continued

Term Definition Comment

Sparkless Ca2�

releaseCa2� release lacking discrete space-time

substructure.See Refs. 32, 33, 230, 232, 332, 440. Resolving subtle local events

depends on both signal contrast and detection sensitivity.Ca2� blink Local depletion of Ca2� in a single ER/SR

cistern during a Ca2� spark.See Ref. 48. Ca2� spark and blink are complementary signals in the

cytosol and the ER/SR, respectively. They represent two views ofthe same elementary event. Synonym to “skraps” in skeletalmuscle.

Ca2� skraps Backward spelling of “sparks.” Name forthe luminal Ca2� depletion signalduring a spark.

See Ref. 213. Synonym to blinks in skeletal muscle.

Ca2� scraps Global or local ER/SR Ca2� depletionduring a large global Ca2� release.

See Ref. 327. Not to be confused with Ca2� blinks or skraps.

Ca2� mark Single-mitochondrial Ca2� transient. See Ref. 285. Seen in H9c2 cardiac cell line. Thought to be locallytriggered by Ca2� sparks but direct evidence is lacking.

Ca2� clock Oscillatory local or global Ca2� releaseactivity. Its coupling to theelectrophysiological clock is made byBKCa, ClCa, and NCX and modulatesmembrane excitability.

See Refs. 208, 391. Its time-keeping mechanism may be intrinsic tothe store Ca2� release or is entangled with the electrophysiologicalclock. In the latter case, the charge of the store by AP-evoked Ca2�

influx sets the SCICR in motion, and the gradual discharge of thestore keeps the time. In pathophysiology, SOICR can make theclock a time bomb to trigger arrhythmias. See Table 3 for details.

Spontaneoustransientinwardcurrent(STIC)

Reflects activation of ClCa by asubsurface Ca2� spark.

See Ref. 166.The magnitude and direction of ClCa current are voltagedependent.

Spontaneoustransientoutwardcurrent(STOC)

Reflects activation of BKCa channels by asubsurface Ca2� spark.

See Refs. 19, 37. Also called spontaneous miniature outward currentor SMOC. The magnitude and direction of BKCa current are voltagedependent.

Spontaneoustransientoutwardthen inwardcurrent(STOIC)

Reflects time-dependent activation ofmixed BKCa and ClCa currents by asubsurface Ca2� spark.

See Ref. 446.

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Ca2� sparks continue to occur when LCCs have beenblocked pharmacologically. Even surface membrane in-tegrity is not a requisite for cardiac spark production,since they are readily observed in saponin-permeabilizedmyocyte preparations (242, 244). It has been concludedthat spontaneous Ca2� sparks are due to the small butfinite opening rate of RyRs that depends on various fac-tors including [Ca2�]i and [Ca2�]SR, the free Ca2� concen-tration in the cytosol and in the SR lumen, respectively.The spontaneous spark rate of 100 per cell per secondsuggests an opening rate of RyR under these conditions of

�10�4 s�1, providing that a typical cell contains 106 RyRs(69). Notably, a recent study placed the rate of Ca2� sparkoccurrence over two orders of magnitude, ranging from�50 to 5,000 per cell per second; its distribution is highlyskewed toward the low-frequency end (11). It should becautioned that freshly isolated cardiac cells displayinghigh Ca2� spark activity are usually less Ca2�-tolerant andare perhaps those that are somewhat damaged during theprocedure of cell isolation.

Ca2� spark rate increases in a concentration-depen-dent manner in the presence of the methylxanthine caf-

TABLE 2. Choice of indicators

As a technical note, fluo 3, fluo 4, rhod 2, and Oregon Green 488 BAPTA-1 have been the indicators of choice in Ca2� spark experiments. Thisis because of their unique combination of superb dynamic range (for fluo 3, �100-fold increase in fluorescence intensity upon Ca2�

association) (160), visible light excitation, appropriate kinetics, and dissociation constant (for fluo 3, Kd � 0.4 �M in physiological saline;�1.1 �M in skeletal muscle cells due to protein binding of the indicator) (159). Collectively, these features confer a large signal-to-background contrast, fast “on” and “off” kinetics, and high sensitivity when the indicator responds to local, short-lived �Ca2�� gradients. Forsimilar reasons, the low-affinity Ca2� indicator fluo 5N (Kd � 135 �M in saline and 400 �M in cells) (327) has been used to measure Ca2�

blinks inside the SR lumen (48). Microscopic SR Ca2� depletion has also been recorded by exploiting the shifted excitation and emissionratioing (SEER) imaging of Mag-indo 1 (213). In contrast, indo 1 and fura 2, two popular Ca2� indicators for ratiometric measurement ofglobal �Ca2��i transients, perform poorly in spark imaging (W. J. Lederer, personal communications).

None of the aforementioned indicators for spark detection displays a significant shift in excitation or emission spectrum upon Ca2� association,which precludes the use of standard ratiometric measurement. In estimating the �Ca2�� involved from the observed fluorescence signal (F), acommon practice has been to express the data as the ratio R � F/F0, where F0 refers to the baseline fluorescence at resting �Ca2�� (�Ca2��0).Assuming that local �Ca2�� and F are in equilibrium and neglecting the fluorescence from the Ca2�-unbound indicator species, we have (69)

�Ca2�� KdR/(Kd/�Ca2��0 �1 �R)With further knowledge of �Ca2��0, one can then estimate the level of �Ca2�� involved. An alternative method to determine �Ca2�� is to measure

the maximal fluorescence at saturating �Ca2�� (Fmax) and determine the �Ca2�� according to the following equation (327)�Ca2�� KdF/(Fmax � F)The second approach is preferred if �Ca2��0 is uncertain or varying. For spike measurement, the choice of indicator would be a low-affinity

indicator (implying fast kinetics and linearity at high local �Ca2��) with good dynamic range, for example, Oregon Green 488 BAPTA 5N orfluo 5N at millimolar concentrations. However, fluo 3 or fluo 4 should also work because diffusive replenishment of Ca2�-free indicator wouldalleviate the problem of saturation.

FIG. 1. Ca2� sparks. A: two-dimensional confocal images of Ca2� sparks in a quiescent cardiac myocyte (scan rate 1.0 s/frame). B: line scanconfocal images of an action potential (AP)-elicited [Ca2�]i transient (top) and a spontaneous spark (bottom) (scan rate 2.0 ms/line). Time and spaceordinates are displayed in the horizontal and vertical directions, respectively. [Modified from Cheng et al. (69) and Cheng and Wang (71).]



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feine, which sensitizes RyRs to Ca2�, or decreases in thepresence of the RyR inhibitor tetracaine (243). The re-sponse usually involves a transient phase because of theself-correcting nature of the process: as the Ca2� sparkrate increases, the efflux of Ca2� from the SR increases.The SR Ca2� depletion produced by the increased Ca2�

spark rate tends to lower the Ca2� spark rate back to-wards its original rate. A high concentration of caffeine(�5 mM) opens all RyRs and thus completely empties theSR, giving rise to a full-fledged [Ca2�]i transient and thenmasquerades as a release inhibitor. Ryanodine itselfcauses complex, concentration-dependent actions. Atsubmicromolar concentrations, it induces repetitive Ca2�

sparks at the same sites, or promotes sustained, low-amplitude Ca2� elevations or “embers” (142) (Table 1)that trail their initial peaks (69). At supramicromolar con-

centrations, ryanodine abolishes sparks altogether. OtherRyR modifiers such as FK506, rapamycin, and Impera-toxin A also evoke trailing embers in a subpopulation ofsparks (242, 375, 420), in qualitative agreement with theplanar lipid bilayer observations.

B. Evoked Sparks

During cardiac EC coupling, Ca2� sparks are evokedby Ca2� influx through LCCs via the Ca2�-induced Ca2�

release (CICR) mechanism (Fig. 2). The evoked sparksappear to be identical to spontaneous ones in terms ofamplitude, kinetics, and spatial properties. Depolarizationper se is ineffective in evoking sparks. For instance, nodepolarization-triggered sparks are seen upon removal of

FIG. 2. Depolarization-evoked Ca2� sparks.Under conditions of whole cell patch clamp andintracellular dialysis of the indicator fluo 3, asmall depolarization (from �50 mV holding po-tential to �40 mV) evoked Ca2� sparks ran-domly in space and time. Summation of thesediscrete, brief, and localized events determinesthe time course and magnitude of the global[Ca2�]i signal (bottom). Scale bar: 10 �m. [Mod-ified from Wang et al. (390).]

FIG. 3. Ca2� spikes. Cardiac myocyte un-der whole cell voltage clamping was dialyzedwith 4 mM EGTA and 1 mM Oregon Green 488BAPTA 5N via the patch pipette, and the con-focal scan line was placed along the celllength, perpendicular to the transverse tubule(TT). A: depolarization to 0 mV evoked dis-crete Ca2� spikes at TTs that were roughly 1.8�m apart. B: spatially averaged fluorescencesignal, which, to the first approximation, isproportional to the SR Ca2� release flux (Jsr).C: contour plot of Ca2� spikes in A. D: surfaceplot of a line scan image from another cell,showing both spontaneous (at the holding po-tential �70 mV) and evoked Ca2� spikes (at�30 mV). (Figure courtesy of L. S. Song.)

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extracellular Ca2�, or replacing it with Ba2� as the chargecarrier, or depolarization beyond the apparent reversalpotential of Ca2� currents (�80 mV). These findings indi-cate that Ca2� entry through LCCs serves as the trigger ofevoked Ca2� sparks in heart.

Individual Ca2� sparks can be resolved only whenthey are activated at a low density, not fusing with eachother in space or time. Accordingly, evoked sparks areseen at the leading edge of a [Ca2�]i transient, with near-threshold activation of LCC, during very brief (ms) depo-larization, with reduced extracellular [Ca2�], or afterpharmacologically reduced LCC availability. In the lattercase, both the residual Ca2� currents (ICa) and the prob-ability of Ca2� spark activation (Pspark) display a bell-shaped voltage dependence. However, the voltage depen-dence of Pspark shifts to more negative voltages than ICa

under these conditions, indicating that the total ICa doesnot uniquely determine the number of evoked sparks(315). The finding is consistent with an earlier observationthat the gain function of EC coupling (G), defined as theratio of whole cell release Ca2� flux over the whole cellICa, decreases at positive potentials (407). It is thoughtthat microscopic properties of ICa such as the amplitudeof unitary LCC current (iLCC) constitute additional deter-minants of EC coupling efficiency (see below).

C. Sparks and EC Coupling

It is now widely accepted that Ca2� sparks constitutethe elementary events of cardiac EC coupling. Stated in aslightly different way, depolarization-triggered [Ca2�]i

transients arise from the stochastic summation of discretesparks. This view is based on two criteria: 1) evokedsparks are “atomic” or indivisible under physiologicalconditions (even though they are splittable under certainexperimental conditions and are not of uniform size, seebelow) and 2) summation of Ca2� sparks can explainprominent features of [Ca2�]i transients. In this regard, ithas been shown that evoked sparks account for the vastmajority, if not the totality, of Ca2� release flux in heartcells (357). Spatial nonuniformity in the rising phase of[Ca2�]i transients can be reconstructed by appropriatelyplacing Ca2� sparks in space and time (58). During full-fledged cardiac EC coupling, �104 sparks are evokedwithin a few tens of milliseconds in a single myocyte,summing into a global [Ca2�]i transient of �1 �M. Thisestimate agrees roughly with the estimated numbers ofCRUs in a typical cell (�104 CRUs each consisting of�102 RyRs) (58, 130), suggesting that most, if not all,CRUs are activated during cardiac EC coupling.

The view that global [Ca2�]i transients consist ofatomic digital units rather than being a continuum hasrevolutionized our understanding of cardiac functionalregulation. Simple, robust, and graded control of cardiac

EC coupling can be achieved by varying the number ofsparks recruited. Specifically, the characteristic bell-shaped voltage dependence of global [Ca2�]i transientscan now be ascribed to a similar voltage dependence ofspark production, while the unitary properties of evokedsparks are voltage independent. Furthermore, many phy-siological modulations of cardiac contractility are nowappreciated to be mediated primarily by altering the mag-nitude and kinetics of spark activation. Examples includecontractile regulation by �-adrenergic receptor (�-AR)stimulation (444), NO production during stretch (294),mitochondrially derived ROS (425), and reactive nitrogenspecies (RNS) (378). Intrinsic variability in unitary sparks,the properties and nature of “sparkless” release, as well asthe “paradox of CICR” (363) are discussed later.

D. Sparks and Ca2� Waves

Spontaneous Ca2� sparks from single CRUs usuallyremain local and solitary despite the CICR mechanismthat operates in ventricular myocytes. Within a sarco-mere, Ca2� sparks tend to center on the transverse tu-bules (TTs) at the Z-disk of a sarcomere, where discreteCRUs are spread on a plane that is perpendicular to thelong axis of the cell, with a nearest-neighbor averagedistance of 0.7 �m in the rat (130, 352). In the roughly 8 flvolume of the Ca2� spark, there are between 2 and 5CRUs (48, 352). It appears that none of these neighboringCRUs engulfed in the volume of a spontaneous Ca2�

spark is activated. Under Ca2� overload conditions (e.g.,high extracellular Ca2�), however, spontaneous Ca2�

sparks do activate nearby CRUs. This is reflected bynearly synchronous activation of multiple sparks fromneighboring CRUs, seen as a “compound spark” or “mac-rospark” (Table 1). Such spark-induced spark activationoccurs more readily in the transverse direction, amongCRUs separated by 0.5–1.5 �m on the same Z-disk, than inthe longitudinal direction, among CRUs on adjacent Z-disks at �1.8 �m intervals (288). A number of factors,including greater spark amplitude and duration, and ele-vated [Ca2�]i and [Ca2�]SR that sensitize CICR, may act ina synergistic fashion to promote spark propagation. Or-derly activation of sparks in space and time then evolveinto a propagating wave of elevated [Ca2�]i. As best seenduring initiation and low-velocity propagation, Ca2�

waves are often triggered by compound sparks, and thenhop forward from one Z-disk to the next (67). Microscopicspiral Ca2� waves (231) also develop in cardiac myocytesdisplaying high spark activity. These observations, to-gether with theoretical modeling (181, 344), support thenotion that Ca2� sparks participate as crucial events inthe initiation and propagation of Ca2� waves.

In neonatal cardiac myocytes, Luo et al. (244) haveshown that perinuclear sparks trigger perinuclear Ca2�



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waves that engulf the entire nucleus without traveling intothe bulk cytosol, affording a mechanism for active nuclearCa2� regulation independent of the bulk cytosolic Ca2�.Specifically, Ca2� sparks arising from both RyRs andIP3Rs are relatively concentrated in the perinuclear re-gion, where immunocytochemical evidence suggests theexistence of a higher release channel density. When theIP3-linked receptor signaling pathway is activated, perinu-clear sparks and waves become more frequent, and inter-mittently generate Ca2� waves that sweep over the entirecytoplasm, indicating that perinuclear sparks and wavesalso act as the pacemaker of global [Ca2�]i oscillations indeveloping cells (244).

E. Sparks in Different Types of Cardiac Cells

To date, Ca2� sparks are found in the ventricularmyocytes of mammals, including rat (69), mouse (68),rabbit (48, 233, 317), ferret (319), guinea pig (239, 240),dog (356), human (134, 226, 241), and bird (H. Cheng,personal communication). Similar Ca2� sparks have alsobeen recorded in isolated cardiac trabeculae (408), heartslices, and intact perfused heart (H. Cheng, personal com-munication). In atrial myocytes, which largely lack TTsand contain both peripheral junctional and central non-junctional CRUs, spontaneous Ca2� sparks are larger andlonger lasting than their ventricular counterparts (300,000Ca2� in 12 ms versus 100,000 Ca2� in 7 ms), with a highprevalence at the periphery (330, 415, 416). The actionpotential (AP) directly evokes subsurface Ca2� sparks,

which then activate inwardly propagating waves of CICR.In Purkinje fiber cells that contain RyR2, RyR3, and IP3Rsorganized in a complex, layered structure, regional differ-ences in the properties of Ca2� sparks and “Ca2� wave-lets” (Table 1) have been documented (368). Rhythmiclocal Ca2� release events, manifested as Ca2� sparks andcompound sparks, are also found in sinoatrial nodal cellsand are thought to cause diastolic depolarization via localactivation of the electrogenic NCX (35, 36), thereby serv-ing as a “Ca2� clock” that is coupled to the “electrophys-iological clock” made up of ionic currents to regulate thepacemaker activity (208, 386, 387) (Table 3). PerinuclearCa2� sparks and waves play an important role in theautonomous regulation of nuclear Ca2� signaling in neo-natal rat cardiac myocytes (244) and embryonic (E18)mouse cardiac myocytes (182). Likewise, robust sparkactivity is detected in cardiac myocytes derived from invitro stem cell differentiation, but is absent after geneticablation of RyR2 (426). In the H9c2 cardiac cell line,spontaneous sparks are thought to trigger single-mito-chondrial [Ca2�] transients (“Ca2� marks”) (Table 1),though direct evidence is lacking (285). Sparklike eventsare also found in cardiac fibroblasts (C. Wei, personalcommunication). Thus spark production is a propertycommon to all types of cardiac cells. Characteristics ofsubcellular Ca2� signaling differ in different types of cells,perhaps matching cell-specific functions. In particular,distinctive perinuclear Ca2� dynamics in embryonic andneonatal cardiac cells may encode information for thetranscriptional regulation of cardiac development.

TABLE 3. Ca2� clock: what makes it tick?

Rhythmic store-mediated Ca2� signals have been demonstrated at multiple levels in many types of cells. Cardiac and skeletal muscle Ca2�

sparks exhibit rather stereotyped duration, as if some time-keeping mechanism or a molecular “clock” is controlling the elementary release(141, 391). At hyperactive spark sites, repetitive firing of spontaneous sparks occurs with quasi-periodicity, and there is a minimal delaybefore the next spark can fire, also suggesting a time-keeping mechanism (391). These results indicate that a CRU and its connected releasemachinery may represent the tiniest supramolecular clock known to date. These findings have come as surprises because they implythermodynamically irreversible gating of CRUs, which, in turn, requires a coupling of the CRU gating to a free energy source (141, 391). Atthe single-channel level, however, planar lipid bilayer recording failed to reveal any preferred RyR open or close duration, suggesting that asingle RyR per se does not act as the clock (391).

The Ca2� clock phenomenon has also been demonstrated at the whole cell level. Rios et al. (308) have demonstrated quasi-periodic dampingoscillatory SR Ca2� releases in voltage-clamped skeletal muscle fibers. In a series of elegant studies, Lakatta and colleagues (208, 386, 387)characterized the Ca2� clock in sinoatrial pacemaker cells of rabbit heart. Subsurface Ca2� release in the form of sparks and compoundsparks occurs during early pacemaking depolarization and, via induction of inward NCX current, accelerates the depolarization. Instead ofbeing a consequence of membrane depolarization, such local release has its own rhythm, damping oscillatory subsurface release persists forup to 10 cycles when the pacemaker cell is voltage-clamped at �80 mV. Thus, in addition to the classic electrophysiological clock made up ofpacemaker ionic currents of the cell, subsurface Ca2� release acts as a Ca2� clock to make the heart tick. Interestingly, the periodicity of theCa2� clock is similar to that of normal pacemaking action potentials. Inhibition of the Ca2� clock will greatly slow down the frequency ofaction potentials, suggesting that the Ca2� clock is the master that enslaves the electrophysiological clock. The time-keeping mechanism ofthe sinoatrial Ca2� clock is currently under investigation.

An equally elegant example is found in ureteric smooth muscle (52). In this case, the Ca2� clock is a slave to the electrophysiological clock,because an action potential activates Ca2� influx to upload the SR to set the Ca2� clock tick, apparently by the SCICR mechanism (Table 6).Its coupling to the electrophysiological clock involves alterations in BKCa conductance (but not hyperpolarizing currents) (see text). Gradualdissipation of the SR Ca2� confers the time-keeping mechanism. (In metaphor, the SR acts like an hourglass!) Functionally, the Ca2� clockworks to slow the tick of the electrophysiological clock, rendering an exceptional long (�70 s) refractory period after discharge of an actionpotential.

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F. Sparks in Other Types of Cells

Microdomains of elevated [Ca2�] resulting from in-tracellular Ca2� release are universal to virtually all celltypes examined. In particular, Ca2� sparks are present inskeletal (16, 168, 197, 381) and smooth muscle myocytes(52, 274, 446), neuroendocrine cells (e.g., chromaffincells) (447), and neurons containing different isoforms ofRyRs (95, 202, 284). Discrete, local Ca2� release eventsmediated by IP3R, termed “Ca2� puffs” (428) and “blips”(289), are well characterized in Xenopus oocytes. LocalCa2� release events with spark characteristics have beenrecorded even in nonexcitable cells, including endothelialcells (172), oligodendrocyte progenitors (156), and HeLacells (40) and are mediated by either RyRs or IP3Rs orboth. See below for discussion of sparkless release.

G. Terminology

The terminology for the ever-growing collection oflocal Ca2� signaling events is summarized in Table 1.Traditionally, the term “spark” is used to describe Ca2�

release events originating from RyRs while “puff” refersto those from IP3Rs only and “blip” for a very small puff.“Spark” has also been used to name an event mediated bya mixture of RyRs and IP3Rs. For uniform nomenclature,we use “spark” as a generic name to describe a local Ca2�

release event from a single CRU regardless of the molec-ular origin or cell type. Other terms stemming from itinclude “sparklet” referring to a single-channel event and“compound spark” referring to an event consisting ofmultiple CRUs. For instance, “IP3R Ca2� spark” is usedinterchangeably with “Ca2� puff” or “Ca2� blip,” depend-ing on the context (Table 1).

IV. THEORY OF SPARK FORMATION

AND DETECTION

A. Spark Formation

Before delving into the details of the spark formationmechanism, we wish to distinguish among the following:1) the underlying event of Ca2� release flux; 2) the localincrease in Ca2� concentration (a local [Ca2�]i transient),in the presence of a dye; 3) the fluorescence transient (afluorescence spark in object space); and 4) the image ofthe fluorescence spark (the true fluorescence sparkblurred by the imaging system). The term Ca2� spark

usually denotes either the underlying local [Ca2�]i tran-sient or the observed fluorescence spark, in a context-sensitive manner.

Theoretically, the formation of a spark is a “diffusion-reaction” process: the released Ca2� from a spatially de-

limited source diffuse in the cytosol, being removed byvarious buffers and transport mechanisms (69, 301, 344).Governed by Fick’s law of diffusion, a sheer [Ca2�]i gra-dient develops around the point source. The vast major-ity of Ca2� (up to 99%), however, are initially bound toa variety of Ca2� binding sites on proteins and lipidmembranes and, in the declining phase of the spark, areliberated from these sites. Ca2� is also transported intothe SR by SERCA2a, extruded from the cytosol throughthe NCX and the sarcolemmal Ca2� ATPase, and takeninto the mitochondria. Among these mechanisms, diffu-sion and buffering dominate in shaping the local [Ca2�]i

transient. In a global [Ca2�]i transient, however, the de-cline of [Ca2�]i is due largely to extrusion and the SRreuptake mechanisms.

Inside the subspace of a “couplon” (Table 1), theimmediate volume into which Ca2� release flux is in-jected, two novel determinants shape the nanoscopic[Ca2�] gradients: the geometric constraints (200–500 nmin diameter, 12–15 nm thick, large fractional volume oc-cupied by the densely packed RyR feet) and the surfacecharge effect on the electrodiffusion of Ca2�. Numericalsimulations by Soeller and Cannell (61, 350) show that theestablishment and dissipation of [Ca2�]subspace gradientsoccur on the time scale of 100 �s upon the “on” or “off” ofCa2� flux, and the magnitude of [Ca2�]subspace dependsnearly linearly on the magnitude of Ca2� current injected.

There is evidence suggesting that diffusion of Ca2�

and indicator in the cytosol should be considered aniso-tropic (i.e., diffusion coefficient differs in different direc-tions). In ventricular myocytes, myofilaments and othercytoskeletal element are arranged in a complex reticularnetwork. Mitochondria, which occupy �35% of the cellvolume, are assembled into two groups: longitudinallyoriented intramyofibrillar mitochondria and shorter androunder perinuclear mitochondria. These structures mayenforce a “channeled” diffusion. If this is the case, it mighthelp to explain the observation that cardiac Ca2� sparkwidth is about double numerical model predictions and is,on average, 20% greater in the longitudinal than the trans-verse direction (68, but see also Ref. 11). An idea ofnon-Fickian diffusion in spark formation has been pro-posed recently (370).

B. Spark Detection

As discussed above, the [Ca2�] gradients due to localrelease flux are first transformed into the [Ca2� indicator]or fluorescence gradients. Wide ranges of imaging tech-niques have been applied to measure this fluorescencesignal , including single-photon (57, 69, 197, 240, 274, 381)and two-photon excitation confocal microscopy (97, 184,291, 351), total internal reflection fluorescence micros-copy (TIRFM) (80), and wide-field microscopy coupled



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with a low-noise CCD camera (446). The generic problemswith optical fluorescent spark detection are twofold. 1) Op-tical blurring is defined by the point spread function (PSF) ofthe imaging system. A PSF depicts the three-dimensional(3D) intensity description of the image produced by a pointobject of unit intensity. For a 3D object (e.g., a fluorescencespark), its image is the convolution of the 3D object with thePSF as the weighing matrix. The sharper the PSF, thesmaller the blurring effect. Confocal microscopy offers asharp PSF, but is never blur-free. A typical confocal PSF (0.4�m by 1.0 �m in the axial and radial directions for sparkimaging) is almost comparable to the size of a spark. 2)Out-of-focus sparks: the observed population of sparks rep-resents a random sampling of sparks at uncertain locations,and there are no established criteria to discriminate in-focusfrom out-of-focus sparks. Owing to random sampling, theamplitude distribution of observed sparks should follow amonotonic decreasing function, regardless of the true sparkamplitude distribution (70, 334).

Another confounding factor for Ca2� spark eventdetection and measurement is related to noise in thedigital fluorescence images. For spark imaging, we aredealing with an extremely low-level fluorescence signalthat is limited by the indicator concentration and theamount of illumination applicable to the cell. It is recog-nized that indicator inclusion perturbs the physiologicalprocess under study, and illumination stimulates photo-chemical reactions (e.g., ROS production) and, when inexcess, causes damage to the cell and photobleaches theindicator. Furthermore, resolving a fast signal from aspatially confined source is demanding in both speed(temporal resolution) and sharpness (spatial resolution).The typical sampling time and volume of a pixel is of theorder of microseconds and femtoliters, respectively, inconfocal spark imaging. Under stringent experimentalconditions, only a few photons can be collected in a pixel.Thus the photon-shot noise intrinsic to fluorescence emis-sion and detection imposes a physical limit on sparkresolution. In practice, there are tradeoffs among theconflicting requirements of signal-to-noise ratio, spatio-temporal resolution, photostimulation, photodamage, andthe indicator perturbation of the [Ca2�]i gradients under

study. With regard to spark image processing, automatedspark detection algorithms are necessary for objectivespark detection in a noisy background (70, 295) (Table 4).

C. Ca2� Spikes

A rather unique experimental setting has been devel-oped to resolve the source location and time course ofCa2� release in a spark (324, 357). This involves an ad-mixture of a fast, low-affinity Ca2� indicator (e.g., OregonGreen 488 BAPTA 5N, 1 mM, Kd � 31 �M) and an excessof high-affinity, but slow Ca2� chelator (e.g., EGTA, 5 mM,Kd � 150 nM at pH 7.2). Owing to the kinetic disparitybetween the indicator and the buffer, newly released Ca2�

ions tend to bind to the indicator before they are capturedby EGTA. The initial ratio of Ca2� binding to the fluores-cent indicator and to EGTA is determined by kon,indicator

[indicator]/kon,EGTA[EGTA]. Since the association rateconstant kon,indicator is typically �100-fold faster thankon,EGTA, EGTA is far less effective in Ca2� binding in theclose vicinity of the release site. As Ca2� ions diffuseaway, however, nearly all of them are eventually capturedby the EGTA in excess. As a result, the fluorescent signalis spatially more restricted than in a regular spark and, inthe limit of a high concentration of Ca2� buffer, closelyfollow the waveform of Ca2� release flux (357). Thesharply defined local Ca2� signals are called “Ca2� spikes”(357) (Table 1), which track the time course and pinpointthe origin of local Ca2� flux. In heart muscle cells, Ca2�

spikes have been used to visualize Ca2� release flux duringspontaneous Ca2� sparks (70) and TT-SR couplon activationduring full-fledged EC coupling (Fig. 3) (32, 314, 324, 357,360). A TT-SR Ca2� spike reflects a single or a compoundCa2� spark, depending on the number of CRUs recruited.Similar approaches have been widely applied in two-dimen-sional (2D) mapping of membrane Ca2� influx in neurons(436), the organization of CRUs in dorsal root ganglion(DRG) cells (283), LCC Ca2� sparklets in smooth musclecells (273), and Ca2� puffs and blips (i.e., IP3R Ca2� sparksand sparklets) in Xenopus oocytes (55).

By the same reasoning that EGTA does not sup-press local indicator signal, inclusion of EGTA up to

TABLE 4. Automated spark detection

Discerning sparks amidst a noisy background is not as straightforward as one might expect. Because of noise, not all sparks registered inimages can be detected (false negatives) and not all detected events are truly sparks (false positives). Early spark analysis relied on eyeballidentification of spark events in the images; the result was subject to observer bias, and inconsistencies were noticed among differentstudies. To provide objective criteria for statistics- and feature-based spark detection, we developed the first automated computer algorithmfor spark detection, implemented it in IDL (Interactive Data Language), and tested its error rates with respect to false positives and falsenegatives (70). Nowadays many variants of the spark detection algorithm in various computer languages have been developed as community-shared resources (11, 44, 295, 397). Objective, precise, and high-throughput measurement of sparks in either linescan (xt) or fast 2D (xy-t)images is a prerequisite for elucidation of spark mechanisms and detection of even subtler changes in different experimental,physiological, and pathophysiological circumstances. Future development of more sensitive, robust and reliable detection algorithmswill likely complement the development of novel Ca2� indicators (379) and imaging techniques (31) to expand the frontiers ofmicrodomain and nanodomain Ca2� imaging.

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5–10 mM should not significantly affect CICR betweenLCCs and RyRs (324, 357, 411) nor the retrograde inac-tivation of LCCs by released Ca2� (323), both occurringon the nanometer scale. Nevertheless, excessive Ca2�

buffering would hamper Ca2� communication on themicrometer scale, such as CICR between adjacentCRUs. In addition, high [EGTA] would “clamp” the bulk[Ca2�]i, suppressing global [Ca2�]i transients and slowingthe refilling of the ER/SR (324, 357, 360).

D. Inferring [Ca2�]i Gradients From Sparks

When we infer local [Ca2�]i gradients from the fluo-rescence signal, it is important to distinguish between[Ca2�]i sparks per se (i.e., the aforementioned micro-scopic, transient [Ca2�]i gradients), true fluorescencesparks (with no optical distortion), and the fluorescencesparks as captured by an optical imaging system. In theCa2�-to-fluorescence transform, indicator saturation in-troduces nonlinearity when [Ca2�]i is well above the Kd ofthe indicator. With regard to the kinetics, the ability of thefluorescent signal to track rapid [Ca2�]i rise is primarilydetermined by Kon,indicator, whereas Koff,indicator serves as alow-pass filter to the dissipation of the fluorescencespark. The diffusion of the Ca2�-bound indicators alsosmears the spatial [Ca2�]i gradients of a developing spark.Numerical simulations confirm that the fluorescence spark,even free of optical blurring, differs drastically from theunderlying [Ca2�]i spark, underestimating the high [Ca2�]i

near the origin and overestimating [Ca2�]i at distant regions,whereas its time-to-peak roughly tracks the duration of Ca2�

flux (assuming a sharp turnoff of the release) (69, 301, 344).The observations that the protein-binding of the indicatoralters Ca2� association and dissociation kinetics (159)should also be taken into account for quantitative under-standing of spark formation and detection.

V. SPARK MORPHOLOGY

The Ca2� spark is a direct measure of the gating ofarrayed RyRs and the resultant microdomain Ca2� inintact cells, providing a real-time report on the behaviorof the CRU and primary information on Ca2� efflux fromthe ER/SR. Spark morphology thus bears rich informationabout CRU gating, release flux, microscopic Ca2� diffu-sion-reaction, and physiological regulation of these pro-cesses. However, tapping the information concealed inspark morphology often requires judicious examinationof various aspects of spark formation and detection.

A. Amplitude

Measured fluorescence spark amplitude varies widelyfrom the lower end set by the detection threshold (�F/F0

�0.2) to the upper end set by the brightest ones (�F/F0

�4 in ventricular myocytes). Confocal sampling theorystates that the apparent spark, which represents an opti-cal section of the true fluorescence spark at randomangles, displays reduced amplitude, broadened spatialspreading, and blunted kinetics, with the degree of dis-tortion depending on the relative positioning between thetrue spark origin and the focal plane or scan line. Regard-less of the true spark amplitude, the apparent spark am-plitude always obeys a monotonically decaying distribu-tion (181, 334), analogous to the situation that more dimthan bright stars are visible to an earth-bound stargazer.Experiments aided by an automated spark detection al-gorithm corroborate this prediction for both cardiac andskeletal muscle sparks (70, 141). In this regard, the aver-age amplitude of the observable population of sparksunderestimates the mean of true sparks. The practice ofusing the brightest sparks as the representative events(141, 179), however, is also biased if sparks are not of onesize. It should be noted that the �F/F0 value also dependson [Ca2�] at F0, which likely varies from cell to cell.

In an effort to restore the true population statistics,Rios et al. (308) extracted the true distribution of amplitudefrom the measured amplitude distribution and showed thatthe true distribution was dispersed, and could be modal.Some investigators have exploited sparks at fixed sites, pro-duced either by hyperactive CRUs or by trains of electricalstimuli with partial inhibition of the LCC, to analyze thevariability in spark amplitude. Considerable event-to-eventvariability was detected but has been largely attributed tophoton shot noise (46). More recently, Wang et al. (390) andShen et al. (331) have used the so-called “loose-seal” patch-clamp technique to activate Ca2� sparks from in-focusCRUs. By pressing a patch pipette (tip diameter �1 �m)against the surface membrane to form a low-resistance(20–50 M) seal, repetitive depolarization can evoke a pop-ulation of Ca2� sparks beneath the patch membrane, free ofout-of-focus sparks. With this rigorous characterization ofspark properties, the true amplitude of cardiac sparks ex-hibits a broad, modal distribution. In addition, polymor-phism of sparks has been demonstrated even for eventsfrom the same CRUs. Similarly, Lipp and Niggli (230) dem-onstrated that Ca2� sparks triggered by local photoreleaseof caged Ca2� are highly variable in amplitude. Collectively,these results suggest a revision of the initial thought thatCa2� sparks are stereotypical.

Computer simulations suggest that spark peak ampli-tude roughly reflects the amount of Ca2� released in thebrief rising phase (63, 179, 301, 344). For long-lastingevents, the plateau amplitude is proportional to the time-averaged release flux (346). In Ca2� spike measurement,however, the spike amplitude is more directly related tothe instantaneous release flux (357).



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B. Width

The spatial width of a spark, usually represented bythe full width at half-maximum (FWHM), is a revealingparameter of microscopic Ca2� diffusion and reaction.Cardiac muscle sparks are of 2.0 �m FWHM when mea-sured at the time of the peak signal. Ca2� spikes obtainedin the presence of 4 mM EGTA suggest an upper limit of0.6 �m for the Ca2� source extension (391). However,nearly all numerical models of spark formation predict aFWHM that is only half of the experimentally observedvalue (63, 179, 186, 344, 351), and this twofold differencein FWHM would translate into an eightfold difference involume or “signal mass” (defined as the integral of thefluorescence increase over the volume of a spark), creat-ing the “spark width paradox” (344). This paradox hasbeen difficult to reconcile by simply varying model pa-rameters (source current, geometry of an extendedsource, Ca2� and indicator diffusion, Ca2� buffering, andCa2� pumping) within their physiological limits (344). Forindicator saturation to render a 2.0-�m wide spark, itwould require an exceptionally large release flux (20–40pA) and predict a spark amplitude of F/F0 �6–7 (179).Soeller and Cannell (351) show that a spatially extendedCa2� source combined with a triangle-shaped waveformof release flux affords a possible solution to the sparkwidth paradox. Sparks in intact frog skeletal fiber are of1.0 �m FWHM that can be fully accounted for by a sparkmodel, whereas the spark width paradox persists in per-meabilized or cut-end skeletal fiber preparations from thesame species (63, 167). Differences in spark width inthese muscle fiber preparations might be attributable todifferent Ca2� buffering or release flux or both.

In a more drastic view, Tan et al. (370) questionedwhether the Fickian diffusion model applies to sparkformation. Fick’s second law of diffusion states that thediffusive flux is proportional to the first order of thederivative of the concentration gradient.

In anomalous or non-Fickian diffusion model, diffu-sion with reaction can be given by

�cr, t)�t

� D�

1r2

�

�r�r2

�1��

�t

��c(r, t)�r� � � f(r, t)

where 0 � � � 1, 0 � � � 2, D� is anomalous diffusionconstant and f is the reaction function represnting Ca2�

sources or sinks. When � � � � 1, the equation is sim-plified into Fick’s law.

It is known as anomalous space superdiffusion orsubdiffusion for 0 � � � 1 or 1 � � � 2, which corre-sponds to long or short jumps of the random walker,respectively. The spark data are best depicted by � � � �1.25, suggesting a subdiffusion model in the milieu of thecytoplasm. The physical basis for this non-Fickian behav-

ior is unclear, but it is thought to reflect the complexmicroscopic and nanoscopic structures and viscoelasticproperties of the cytoplasm.

C. Kinetics

The average time to peak of a spark is �5 and 10 msfor skeletal muscle and cardiac sparks, respectively; thehalf decay time is �20 ms in either case (69, 197, 206,391). The brevity of Ca2� sparks ensures the temporallocality of Ca2� signaling and implicates a powerful spark-termination mechanism (see below).

Detailed analysis of the spark release duration, in-dexed by the rise-time of sparks or duration of spikes,uncovered a modal distribution in both skeletal musclefibers and cardiac myocytes (308, 391). This is not ex-pected, if the underlying channel or channel group isgoverned by a Markovian gating mechanism and is freefrom external free energy. The expected open or burstingtime distribution should be the sum of decaying exponen-tials, as required by thermodynamic laws of microscopicreversibility. It was then suggested that the preferred re-lease duration thus necessitates a coupling of the releasechannels to a free energy source (304, 391). In this regard,the rise of [Ca2�]subspace as well as the fall of [Ca2�]SR duringa spark could link RyR gating to the chemical potential of[Ca2�] across the SR membrane. Notably, an inverse rela-tionship between the release duration and the rising rate hasbeen observed (206, 392), consistent with either a[Ca2�]subspace-mediated inactivation or a local store deple-tion-mediated spark termination (see below).

In the presence of reagents that stabilize RyR open-ing at subconductance (e.g., submicromolar ryanodine) orfull conductance (e.g., Imperatoxin A for type 1 RyRs), asubpopulation of Ca2� sparks may last for hundreds ofmilliseconds or even longer (69, 141, 142, 420). Such along-lasting spark usually displays a brief normal peakfollowed by a plateau at reduced amplitude, dubbed atrailing “Ca2� ember” (142). Embers without the initialpeak (or lone embers) have been seen in mammalianadult skeletal muscle (440), and 10-s-long “Ca2� glows” ofregular amplitude (Table 1) have recently been observedin superior cervical ganglion neurons (427). The nonde-caying plateau suggests that the release and replenish-ment of local ER/SR Ca2� quickly reach a balance. Thereplenishment mechanisms consist of local cytosolicCa2� reuptake and, more importantly, luminal Ca2� trans-fer within the connected ER/SR network.

D. Autonomy

Several lines of evidence indicate the autonomy ofthe Ca2� spark: unitary Ca2� spark properties are inde-pendent of the trigger. As the first clue, it has been dem-onstrated that AP-evoked sparks are morphologically in-

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distinguishable from spontaneous sparks (58, 65). A closeexamination revealed that unitary properties of evokedsparks remain constant over the physiological voltagesfrom �50 to �30 mV, despite gross changes of single-channel LCC currents (240). Properties of evoked Ca2�

sparks are also invariant regardless of their latency rela-tive to the onset of the voltage pulse (240). Furthermore,abrupt turnoff of the LCC Ca2� current by hyperpolariza-tion after a 2 ms depolarization does not curtail Ca2�

release in a spark. Conversely, prolonging LCC meanopen time with FPL64176 increases neither spark ampli-tude nor duration (390). Thus, once activated, the evolu-tion of cardiac sparks is no longer controlled by thetrigger Ca2� signal; rather, it appears to be highly auton-omous, determined by properties intrinsic to the CRU andits connected ER/SR cistern.

The polymorphism of Ca2� sparks mentioned aboveshould not be taken as contradictory to the autonomy ofspark production. Rather, it reflects stochastic gating ofCRUs. With exceptions, pharmacological and physiologi-cal modulation of sparks is mainly achieved by alteringthe propensity of Ca2� sparks. This is particularly thecase for RyR activators (low or moderate concentrationsof caffeine, ROS) (51, 197, 319, 378), whereas a RyRinhibitor (e.g., tetracaine) reduces the spark amplitude(390) as if it reduces the effective size of a CRU. The sparkautonomy is not absolute in skeletal muscle where CRUsare physically linked to the voltage sensor and cannot runfreely (see below). Taken together, the autonomy of sparkgenesis strongly supports the notion that sparks are thebuilding blocks of global [Ca2�]i transients.

VI. Ca2� SPARKLETS

Ca2� passing through a single Ca2�-permeant chan-nel opening create a Ca2� microdomain that is fundamen-tal to the architecture of intracellular Ca2� signaling. Thefeasibility of optical recording of single-channel Ca2� mi-crodomains has now been convincingly established for

many types of Ca2�-permeant channels, including L- andN-type Ca2� channels, stretch-activated cation channels,acetylcholine receptor channels, RyRs, and IP3Rs. Com-bining confocal microscopy with the cell-attached patch-clamp technique, Wang et al. (390) first resolved “Ca2�

sparklets,” microscopic [Ca2�]i transients at the mouth ofsingle LCCs in heart cells (Fig. 4). This was done with theaid of the channel agonist FPL64176 to prolong the chan-nel open duration and high extracellular Ca2� to increaseiLCC amplitude. Simultaneous recording of iLCC indicatesthat the amount of Ca2� entry correlates linearly with thetotal fluorescence of a Ca2� sparklet seen in the line scanimage, suggesting that the sparklet provides an “opticalyardstick” for calibration of the local Ca2� signal.

Using a wide-field imaging system equipped with ahigh-speed, low-noise CCD camera, Zou et al. (449) inde-pendently measured Ca2� sparklets arising from theopenings of caffeine-sensitive cation channels andstretch-activated Ca2�-permeant channels in smooth mus-cle myocytes. The 2D images acquired in their experi-ments facilitate spatial integration of the fluorescencesignal to determine the signal mass at the cost of spatialand temporal resolution. Recently, Peng et al. (290) de-veloped an optical bilayer system that visualizes Ca2� fluxwhile recording the unitary current of single RyRs inplanar lipid bilayers. Using 50 mM Ca2� as the chargecarrier and the RyR agonist suramin to stabilize the chan-nel open state (Po �1), they detected bilayer Ca2� spar-klets from reconstituted RyRs carrying 0.25–14 pA Ca2�

current (controlled by varying the transmembrane volt-age). Furthermore, Demuro and Parker recorded Ca2�

sparklets produced by individual N-type voltage-gatedCa2� channels expressed in Xenopus oocytes (100) andindividual muscle acetylcholine receptor channels (98,99). Santana and colleagues (5, 272, 273) combined spikemeasurement and TIRF microscopy to map LCC channelactivity in resting smooth muscle myocytes from rat ce-rebral arteries. They were able to track rare active sites(1–2 in a cell) which display persistent high Po (termed

FIG. 4. Ca2� sparklets. Simultaneousrecordings of single L-type Ca2� channel(LCC) Ca2� current (iCa) and local Ca2�

changes (Ca2� sparklets) at the mouth ofa channel. The ER/SR Ca2� release wasdisabled by thapsigargin and caffeine in acardiac cell, while the iCa was enhancedby inclusion of high Ca2� and the LCCagonist FPL64176 (FPL) in the filling so-lution of the cell-attached patch pipette.[Modified from Wang et al. (390).]



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constitutively active LCCs, nPo �1.25). Up to seven levelsof sparklet amplitude can be resolved at hyperactive sites,affording a visual demonstration of LCC clustering. Inhib-iting or activating the protein kinase C (PKC) pathwaysuppresses or promotes the constitutive activity, suggest-ing that PKC switches the transition of LCC betweendifferent gating modes. These advances show that a newmodality for single-channel recording, single-channel op-tophysiology, extends the horizon of single-channel elec-trophysiology (see Table 5 for further discussion).

VII. INTERMOLECULAR SIGNALING BETWEEN

LCCs AND RYRs

The structural and functional units of cardiac ECcoupling are couplons, which represent supramolecularnanometer-sized assemblies consisting of LCCs on theplasma membrane, arrayed RyRs on the SR cistern, and a12-nm-wide subspace between the apposed membranes(Table 1). Translation of membrane excitation into anintracellular Ca2� signal is mediated by intermolecularcommunication between the two types of Ca2� channelsinside the nanoscopic subspace. In this section, we dis-cuss the fidelity, kinetics, and stoichiometry of LCC-to-RyR coupling.

A. Triggering Sparks by Single LCC Openings

Voltage-dependent spark production in rat ventricu-lar myocytes has been quantified at voltages near the

threshold of LCC activation by using a voltage ramp pro-tocol (from �70 to �40 mV) (57) or small depolarizingsteps (2 mV increments from a �60 mV holding potential)(315). The threshold voltage for spark production is about�55 mV, and the spark probability increases e-fold per 7.5mV depolarization. Both the threshold voltage and thevoltage dependence of spark activation are thereforeidentical to those for LCC activation. Under conditionswhen the LCC is largely blocked, Lopez-Lopez et al.(240) first resolved individual sparks over the entirephysiological voltage range (�50 mV to �40 mV), andat all voltages tested, the time course of spark produc-tion overlaps the time course of the residual ICa. Thenearly identical voltage dependence (over the narrowthreshold voltage range) and the similarities in kinetics(at a given voltage) suggest a linear relationship be-tween LCC activation and spark production. These re-sults indicate that spark activation can be the result ofa single LCC opening (57, 240, 315). The rationale isthat, if spark activation requires the simultaneous acti-vation of n independently gated LCCs, then Pspark

should be the power function of the probability of LCCactivation (PLCC) with exponent n; that is

Pspark � PLCCn

The aforementioned results suggest n �1 under therespective experimental conditions. It should be noted

TABLE 5. Optical single-channel recording

As a celebrated achievement, the introduction of the patch-clamp technique in the early 1980s (158) revolutionized our understanding of ionchannel function by allowing measurement of the ionic currents through a single channel at unprecedented precision. As illustrated in thetext, modern optics, photonics, and the use of ion-selective indicators have extended the horizon by affording a new modality for single Ca2�

channel recording: single-channel optophysiology.A fundamental distinction between optophysiology and electrophysiology resides in the physical principles employed. A single Ca2� ion moving

through a channel contributes two positive electron charges to the electrical current, whereas a Ca2�-bound fluorochrome can emit 105

photons per millisecond at saturating excitation. Even though only a small fraction of these photons can be harvested for readout, thisrepresents a staggering signal amplification which is yet to be fully exploited.

Compared with electrophysiological recording, the optophysiological approach has unique advantages, complementary features, and limitations.First, optical single-channel recording is penetrative, applicable to channels inaccessible to electrophysiological means (e.g., those in TTs,intact tissues, or even in vivo). Second, it is mechanically nonintrusive. As such, it could enable recording of single channels on delicatemembrane structures, channel array assemblies, and macromolecular signaling complexes, leaving the intermolecular interactionsunperturbed. Third, optophysiology. Investigation of channel activity is capable of parallel readout, while retaining the spatial resolution ofsingle-channel events. For instance, a line scan of a cardiac cell can survey �104 RyRs from �100 CRUs at once, and fast 2D imaging maysurvey 10 times more channels simultaneously. This will enable the capture of rare, yet biologically relevant events (e.g., cyclic sparkactivation at fixed sites, and PKC modification of individual channel clusters). Last but not least, the ion selectivity of optophysiology isadvantageous in discriminating among different ion species passing through the channel. This feature has been successfully exploited tomeasure the fractional Ca2� flux through nonselective Ca2�-permeant channels, at the whole cell or single-channel level (434, 445).

At present, the limiting factor for optophysiology is insufficient temporal resolution set and sensitivity by the reaction and diffusion kinetics ofmicrodomain Ca2� and the indicator. For confocal detection, the tiniest Ca2� sparklets resolved thus far consist of �8,000 Ca2� ions,equivalent to a single-channel current of 1 pA lasting 2.5 ms (390). The temporal resolution achieved in another study was �10 ms at best,judged from a 20 ms decay time of sparks. The limited temporal resolution has hampered efforts to resolve LCC Ca2� sparklets underphysiological conditions with unitary current consisting of a mean packet of �110 Ca2� ions. Nevertheless, Zhou et al. (441) have shown thattemporal resolution above that predicted by the indicator’s kinetics can be gained by procedures that entail differentiation of the signal massof a spark, at the price of increased noise. Future technical innovations, particularly in the area of photon collection efficiency, are needed tobring out the full potential of single-channel optophysiology.

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that an n value greater than unity is found when PLCC ishigh but iLCC is small (174).

An unequivocal demonstration that an opening of asingle LCC can indeed trigger a spark came later withcombined cell-attached patch clamping and confocal mi-croscopy. Typical sparks are activated beneath the patchmembrane during an LCC opening under gigaseal patch-clamp conditions (390). However, this is an extremelychallenging experiment; and for routine recording of in-termolecular signaling events, a loose-seal patch-clamptechnique is employed. As shown in Figure 5, LCC spar-klets (modified by FPL64176) can be recorded along withRyR sparks beneath the patch membrane. The two signalsare distinguishable by amplitude and by pharmacologicalmeans. Nearly every spark arises on top of a discerniblesparklet, while 30% of sparklets do not trigger any sparks(Fig. 5), indicating a sparklet-spark coupling fidelity (�) of0.7 in the presence of the LCC modifier.

B. Power Law of Spark Activation

Santana et al. (315) investigated the relationship be-tween trigger [Ca2�] and spark production by controllingiLCC and thereby [Ca2�]subspace, at varying voltages. Qual-itatively, the same ICa does not uniquely determine thenumber of evoked sparks: more sparks are evoked atnegative rather than positive voltages for a given amountof ICa, supporting the idea that the microscopic prop-erties of ICa are also important determinants of ECcoupling (411). Quantitatively, the voltage dependencecurve for the spark-triggering efficiency of ICa (G �Pspark/ICa) closely resembles that of iLCC as depicted bythe Nernst-Planck equation, that is

G � Pspark/ICa � iLCC and ICa � NPLCCiLCC

where N refers to the total number of LCCs in a cell.Therefore

Pspark � NPLCCiLCC2

Given the linear relationship between [Ca2�]subspace

and iLCC (350), we have

Pspark � �Ca2��subspace2

That is, spark activation is a quadratic function of[Ca2�]subspace, requiring the cooperative action of the trig-ger Ca2�. Similarly, a power function with the powerbeing 2 or higher has been reported for RyRs respondingto [Ca2�] step jumps or brief [Ca2�] pulses in planar lipidbilayer experiments (152, 435).

C. Kinetics of Sparklet-Spark Coupling

Simultaneous visualization of sparklets and sparksenabled us to determine the kinetics of intermolecularcoupling between LCCs and RyRs. The latency from theonset of the sparklet to the ignition of the spark varieswidely; its histogram is well depicted by a single expo-nential function with a mean value �6.7 ms, suggestingthat the LCC-to-RyR coupling is a stochastic process of

FIG. 5. Sparklet-spark coupling. A: LCC sparklets (the low-ampli-tude events) and triggered sparks (the high-amplitude events) recordedunder loose-seal patch-clamp conditions. Note that not every sparkletcan trigger a spark. B: histogram of the latency of the sparklet-sparkcoupling and its monoexponential fit ( � 6.7 ms). Data were obtainedin the presence of the LCC agonist FPL64176. [Modified from Wang et al.(390).]



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first-order kinetics. To determine the latency and fidelityunder physiological conditions, we need to develop sometheoretical considerations. If LCC of first-order kineticsdisplays a mean open time LCC, the closing rate constantis 1/LCC. When a spark is activated by a sparklet, thesparklet is interrupted at a rate constant of 1/latency, ,where latency, is the latency of spark activation when anLCC opens indefinitely. Let latency,apparent denote the spar-klet duration; the sparklet termination rate constant(1/latency,apparent) is therefore the sum of the two rateconstants governing the closure of LCC and the triggeringof a spark, respectively

1/latency,apparent � 1/LCC 1/latency,

For the coupling fidelity �, defined as the fraction ofopenings that do trigger a spark, we have

� � LCC/LCC latency, �

With an iLCC of 0.3 pA at 0 mV in the presence ofFPL64176 and high Ca2� (390), the data fit roughly withlatency,apparent �6 ms, LCC �16 ms, latency, �9.6 ms,and � �0.63.

What insights can we gain about LCC-to-RyR cou-pling under physiological conditions? For a LCC of 0.5 msand iLCC of 0.1 pA at 0 mV and 1 mM Ca2� in the absenceof an LCC modifier (149), we estimate that latency, �86ms (by applying the power law of spark activation),latency,apparent �0.5 ms, and � �0.006. A rough estimate of� can also be inferred from the ratio between macroscopic(G, whole cell) and microscopic (�, single CRU) gains ofEC coupling. The latter refers to the total Ca2� flux in aspark relative to that in a sparklet. For spark Ca2� flux(Ispark) of 3 pA and release flux duration of 10 ms, the �value is �600 when a sparklet does trigger a spark, and� � 0 when it does not. Given that G �10 at 0 mV, it canbe deduced that, on average, only 1 out of 60 LCC open-ings can trigger a spark or � � 0.016 at 0 mV, which iswithin threefold of those derived from the sparklet-sparkcoupling. From these two independent approaches, weconclude that many LCC openings trigger a spark at 0 mVunder physiological conditions. There is thus a teleologi-cal need for multiple LCCs to cluster over a 0.2 �m2 dyad(assuming a cisternal diameter of 500 nm) and directdemonstration of this awaits future investigation.

VIII. Ca2� BLINKS

The ER/SR membrane encloses one continuousspace that occupies only a few percent of the cell volume,but is extended throughout the cytosol as interconnectedcisternae and tubules in heart cells (48, 287, 418). Inaddition to serving as the Ca2� storage and release or-

ganelle, increasing evidence indicates that the ER/SRCa2� store actively regulates many Ca2� signaling pro-cesses, such as store-operated Ca2� entry (23, 43, 227,303) and ER/SR stress-mediated cell death (271, 322). Instriated muscles, the SR is organized into two distinctdomains: the free (fSR) and the junctional SR (jSR); thelatter bears the CRUs and contains the low-affinity, high-capacity Ca2� buffer calsequestrin (CSQ) in the cisternae(126, 127).

A. In Cardiac Myocytes

Visualization of Ca2� dynamics in the intricate ER/SRspace has been made possible by the development of anempirical method to load low-affinity Ca2� indicators(e.g., fluo 5N) preferentially into the ER/SR (287, 327).Furthermore, the ER/SR and the cytosol, while overlap-ping under an optical microscope, can be distinguishedexperimentally by targeting spectrally distinctive indica-tors into the two conjugate spaces (48). Building on these,the local jSR Ca2� change during a spark has been inves-tigated along with cytosolic Ca2� measurement. Briefdarkenings of the fluo 5N signal, called “Ca2� blinks” (Fig.6), accompany the production of spontaneous or evokedCa2� sparks in rabbit (48), rat, and mouse ventricularmyocytes (D. X. P. Brochet, personal communication).Similar Ca2� blinks are evoked by APs in the presence ofan LCC inhibitor (to allow for activation of only a fewCRUs). Compared with sparks, blinks are much moreconfined, displaying a typical FWHM of 0.8 �m. Given thesize of a pancake-shaped SR cistern (thickness �30 nm;diameter �465 nm, wrapping around a TT of 100–200 nmdiameter), this observation indicates that Ca2� depletionduring a spark is largely confined to a single cistern,supporting the idea that a spark arises from a single CRU.The magnitude of jSR depletion is substantial, approach-ing 85% of that during a full-fledged global Ca2� release(30–50% depletion). The existence of a jSR-fSR [Ca2�]SR

gradient implies some diffusion restriction connecting thefSR and the jSR. Ultrastructural study reveals that a cis-tern communicates with the fSR network via only one ora few 30 nm diameter tubules at the outer edge of the jSRcistern (48). The rate of Ca2� refilling via these narrowconnections is �35 s�1, as inferred from the blink recoverkinetics (omitting the somewhat 6 times slower reuptakeby SERCA). The presence of Ca2� blinks vividly demon-strates the locality of the ER/SR luminal Ca2� signalingand reveals a new dimension of Ca2� signaling specificityand diversity.

Thus the elementary events of cardiac EC couplingconsist of the trio, Ca2� sparklets, Ca2� sparks, and Ca2�

blinks (Fig. 7). The LCC sparklets activate the spark-blinkpairs via the CICR mechanism operating over a nanome-ter distance. The sparks and blinks are in fact two views

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of the same elementary release event, from the stand-points of the cytosol and the SR lumen, respectively.Summation of sparks gives rise to the [Ca2�]i transient,while summation of blinks results in a negative [Ca2�]SR

transient, known also as “Ca2� scraps” (327). While thesparks and cytosolic Ca2� transients have been under themost scrutiny, it should be emphasized that the blinks andscraps initiate signal transduction on their own (e.g., re-lease termination).

B. In Skeletal Muscle Cells

In saponin-permeabilized amphibian skeletal mus-cle, store depletion in the spark, dubbed “skraps” (213)(Table 1), has also been visualized, but with a numberof surprises. Launikonis et al. (213) developed a shift

excitation and emission ratioing (SEER) method of Magindo 1 fluorescence imaging to measure [Ca2�]SR whenmitochondrial Ca2� uptake is suppressed to limit thecontaminating mitochondrial fluorescent signal. Theirbasic observation is that local [Ca2�]SR depletes by�7% during a spark, comparable to the global SR de-pletion during a twitch (8 –15%). The lower magnitudeof SR depletion in skeletal versus cardiac muscle(�30%) reflects a skeletal SR with larger terminal cis-ternae containing a high concentration of a CSQ withgreater Ca2� binding capacity, thus constituting a muchgreater Ca2� reservoir. The first surprise is that, afterthe brief peak (�10 ms) of the spark, local [Ca2�]SR

continues to decline for nearly 50 ms, apparently vio-lating the law of mass conservation. More surprisingly,a positive [Ca2�]SR transient develops during a cell-

FIG. 6. Ca2� blinks. A: Ca2� blink mirrors Ca2� spark. Data were obtained in an intact rabbit ventricular myocyte whose SR lumen and cytosolic spacewere dual stained with fluo 5N and rhod 2, respectively. Line scan images are presented as F/F0. Left inset: SR was stained more intensely at the TT-SRjunctions (jSR) by fluo 5N, due to greater local SR volume. B and C: time courses (B) and spatial profiles (C) of the spark-blink pair. (Figure courtesy ofD. X. P. Brochet.) D: a typical Ca2� blink rendered in surface plot. [From Brochet et al. (48), copyright 2005 National Academy of Sciences, USA.]

FIG. 7. Sparklet-spark-blink trilogy of cardiacEC coupling. [From Brochet et al. (48), copyright2005 National Academy of Sciences, USA.]



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wide Ca2� release initiated by caffeine or a low [Ca2�]and low [Mg2�] solution. The positive [Ca2�]SR tran-sient is most prominent under moderate stimuli and isoften preceded by a brief decrease in [Ca2�]SR. Theseparadoxical observations prompted a model in whichCSQ depolymerizes upon local release, liberating itsbound Ca2� and giving rise to the positive [Ca2�]SR

transient; after cessation of release, repolymerizationof CSQ accounts for the anomalous kinetics of theskraps; there is no violation of mass conservation if theCSQ-bound Ca2� pool is also taken into account.

IX. SPARK MECHANISMS: ACTIVATION

In this and the next two sections, we discuss sparkmechanisms. What activates a spark? How many RyRsopen in a spark? If more than one RyR is involved, howdo different RyRs coordinate in a spark? What termi-nates a spark? Does a spark impart any after-effect onsubsequent spark activation? See Table 6 for a range ofmechanisms pertinent to sparkology. It should be notedthat not all of them are fully expressed in any given cell,though many may coexist. Their expression and rela-tive dominance depends on the type of cell, its devel-opmental stage, and the subcellular regions of concern(e.g., perinuclear versus subsurface CRUs). We beginby focusing on the mechanisms that trigger a spark (seealso Table 7).

A. Spontaneous Sparks

Spontaneous sparks occur in quiescent unstimulatedventricular myocytes and persist upon transient removal ofextracellular Ca2� (69), blockade of LCCs (58), or chemicalpermeabilization of the surface membrane (242). They areunlikely to be due to Ca2�-indpendent or “constitutive” ac-tivity (Table 6), because RyRs reconstituted in planar lipidbilayers do not display any significant channel activity atsubnanomolar [Ca2�] (121, 152). Instead, they could simplyreflect the probabilistic opening of RyRs at diastolic [Ca2�]i

of �100 nM, via the CICR mechanism. Other trigger signalsalso influence spontaneous Ca2� spark production. Zhanget al. (437) reported that �50% of spontaneous sparks aresensitive to Cd2� blockade and are therefore attributable totriggering by the spontaneous and infrequent openings ofLCCs at resting membrane potential (Vm), in contrast toKatoh et al. (192).

In saponin-permeabilized cells, the DHPR antago-nists nifedipine, nimodipine, FS-2, and calciseptine de-crease spark frequency (88), while the DHPR agonistsBAY K 8644 and FPL-64176 increase it (88, 192, 318),independently of Ca2� entry. Furthermore, the calmodu-lin-binding LA motif of the �1C COOH-terminal tail in-creases spark frequency in the periphery of atrial cells

(417), while the �1C II-III loop peptide inhibits restingCa2� sparks in ferret ventricular myocytes (224). Theseobservations can be accounted for by physical couplingbetween DHPR and RyRs, a mechanism vestigial in heartbut fully expressed in skeletal muscle (Table 6).

RyR activity varies as a function of [Ca2�]SR (150,152, 422), perhaps through altering local [Ca2�]i at themouth of the channel pore (422), directly acting on RyRfrom the luminal side, or interacting with luminal Ca2�

binding proteins and both junctin and triadin in the SRmembrane (151, 154). This “store Ca2�-induced Ca2� re-lease” (SCICR) (Table 6) mechanism may be involved insmooth muscle cells under physiological conditions (52,84, 113). In cardiac cells, however, during time- and thap-sigargin concentration-dependent SR Ca2� depletion,Ca2� sparks become smaller but their rate of occurrenceremains constant if the small-amplitude events missedfrom detection are accounted for (357). This suggests thatlower than physiological [Ca2�]SR does not affect Ca2�

spark production. In contrast, one striking manifestationof SCICR is the so-called “store overload-induced Ca2�

release” (SOICR) (185), whereby [Ca2�]SR reaching athreshold “triggers” sudden and near-synchronous globalSR release (67, 150, 243, 346). The SOICR mechanism maybe particularly relevant to spontaneous spark productionunder Ca2� overload conditions. Both Ca2� spark fre-quency and amplitude increase with elevated extracellu-lar Ca2�, so rogue Ca2� sparks can now trigger neighbor-ing CRUs to form compound sparks and sometimes initi-ate a propagating Ca2� wave (67, 69, 316). Nonetheless,there is no direct evidence to support the hypothesis thatspontaneous Ca2� sparks arise only when a specific SRcistern becomes “overloaded,” as suggested in the litera-ture (185).

Ample evidence shows that the Ca2� spark rate canincrease or decrease at a constant SR Ca2� load by theapplication of agents that sensitize or inhibit RyR activity,respectively; a spark can also progress into an ember inthe presence of RyR modifiers that increase the channelopen duration (see above). For instance, caffeine, bysensitizing CICR, increases spark production in a dose-dependent manner (319) and may cause apparent sparkinhibition by emptying the SR store at high concentrations(10 mM or higher). A more potent and powerful sparkactivator is perhaps nitroxyl. The nitroxyl donor, Angeli’ssalt, dose-dependently increases Ca2� spark frequency incardiac myocytes within minutes of addition. At 1 mM, itcauses an 18-fold increase in the rate of occurrence ofCa

2�

sparks without affecting unitary spark properties(378). Step increase of [Ca2�]i produced by photoreleaseof caged Ca2� evokes Ca2� sparks of variable sizes, inde-pendent of membrane depolarization (230).

Bidirectional regulation of Ca2� spark activity bymitochondrially derived ROS has been observed in intactcardiac myocytes (425). In both cardiac and skeletal mus-

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TABLE 6. Spark mechanisms

Local controltheory

In heart, this refers to the idea that the proximityof LCC to RyR2 clusters can enable a high-gainCICR signaling mechanism to operate withrobust stability.

See Refs. 279, 363. Described and modeled to explain how high-gainpositive feedback Ca2� signaling works in heart. See below.

Ca2�-induced Ca2�

release (CICR)A mechanism whereby an increase in �Ca2��i

elicits Ca2� release from the ER/SR.See Refs. 112, 116, 124. Thought to be the primary method by which

RyRs are triggered to produce Ca2� sparks in heart cells. BothRyR and IP3R use CICR, but IP3R activation also requires thepresence of the coagonist IP3 (21). See text for discussion on theparadox of CICR.

IP3-induced Ca2�

release (IICR)The second messenger IP3 elicits Ca2� release

mediated by IP3Rs in the ER/SR.See Ref. 21. A unifying view of CICR and IICR is that IP3 removes

Ca2�-dependent inhibition of the release channel, and IICR ismerely a manifestation of the shift in CICR sensitivity. See Refs.125, 246.

Depolarization-induced Ca2�

release (DICR)

Depolarization triggers release from the ER/SRindependently of Ca2� entry from the exteriorof the cell.

See Refs. 307, 320. In skeletal muscle, DHPRs serve as the voltagesensors that activate RyRs through conformational coupling. Inheart cells, this is likely a vestigial mechanism accounting for theCa2� entry-independent effect of DHPR ligands on Ca2� sparks(88, 319). In neurons, the underlying molecular mechanism iscurrently unknown (95).

Store Ca2�-induced Ca2�

release (SCICR)

Ca2� loaded in the lumen of the ER/SR activatesthe release channels directly or indirectly bysensitization of CICR on the cytosolic side.

See Refs. 111, 150, 151, 185, 347. One manifestation is store overload-induced release (SOICR) in cardiac myocytes. See Refs. 52, 84, 113for demonstration of robust store Ca2� regulation of the rate ofCa2� sparks in smooth muscle myocytes.

Stretch-inducedCa2� release(SICR)

Membrane tension of the cell causes Ca2�

release from the ER/SR.See Refs. 8, 183, 294. May contain a Ca2� entry-independent

component. Production of second messenger such as NO may beinvolved in some cases (294).

Coupled gating Two or more physically linked channels thatinfluence mutual gating behavior throughmechanical contact.

See Refs. 249, 251. This is seen broadly as a mechanism ofcooperative gating and may thus affect both the opening andclosing of channels that are coupled. It may coexist with CICR.

Constitutivereceptoractivation

A ligand-gated receptor channel manifests a low-level activity in the absence of any ligandoccupant of the receptor.

See Ref. 39 for an example of GPCR signaling and Ref. 444 in thecontext of spark regulation. In planar lipid bilayers, neither RyRnor IP3R displays any significant constitutive channel activity.

Voltage sensordeactivation

Deactivation of voltage sensor in skeletal musclecells enforces early termination of an ongoingspark

See Ref. 206.

Voltage sensorrepression

By physical constraints, DHPR in the nonactiveconformational state prohibits its associationwith RyR from participating in CICR.

See Refs. 333, 440, 443. DICR, DHPR-dependent repression, andvoltage-dependent deactivation may be manifestations of the samemechanism. It helps to explain the lack of Ca2� sparks (335), thepresence of Ca2� embers (91), and the reappearance of ghostsparks in various experimental and pathophysiological conditionsin adult mammalian skeletal muscle. See Ref. 394 for a review.

Stochastic attrition Random closure of independently gated channelsconfers a finite probability for all channels in acluster to close at once.

See Ref. 363. This mechanism could provide a closure mechanismfor a small channel group.

Desensitization ofCICR by storeCa2� depletion

SCICR operating in the reverse mode. See SCICR. This mechanism may contribute to the termination of aCa2� spark via desensitization of CICR during luminal Ca2�

depletion.Channel

inactivationA state in which the channel closes and no

longer responds to stimuli that usually openthe channel.

RyR inactivation can be Ca2� dependent or Ca2� independent. A“fateful” inactivation (296) refers to the notion that all activatedchannels inevitably transit to the inactivation state, in a Ca2�-independent manner. In heart, no experimental evidence suggeststhat any RyR inactivation mechanism occurs rapidly (10 ms orless), but a slow (100 s of ms to seconds) and complicatedinactivation mechanism (see adaptation below) has been observed.

Channeladaptation

A single channel manifests time-dependent decayof Po during a sustained stimulus but, differingfrom inactivation, the channel retains theability to respond to a new, greater stimulus.

See Refs. 66, 152, 383. Channel adaptation can be explained bycomplex schemes of Markovian activation and inactivation.

Quantal release In response to an IP3 signal, there is a transientrelease and then the release stops completely.Yet, subsequent greater IP3 signal can stillelicit yet another transient. The amount of therelease is graded by the intensity of the IP3

signal.

See Refs. 120, 264, 380. By the spike measurement, it is shown to bea property intrinsic to CRU (55). Though it is reminiscent ofchannel adaptation, quantal release has not yet been demonstratedat the single-channel level.



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cles, chemical permeabilization of the cell greatly en-hances spontaneous Ca2� spark production (175, 176,242, 244), a phenomenon thought to be related to alteredmitochondrial ROS metabolism (175, 176). Finally, in thecase of skeletal muscle, removal of DHPR or Mg2� inhi-bition of RyR1 permits the de novo appearance of spon-taneous Ca2� sparks in mammals and augments sparkproduction in amphibians (see below).

B. Triggered Sparks

In cardiac myocytes, sparks evoked during EC cou-pling are triggered predominantly, if not exclusively, byCa2� entry through LCCs via the CICR mechanism. Depo-larization in the absence of extracellular Ca2�, replace-ment of Ca2� with Ba2�, or depolarization to high volt-ages beyond the apparent reversal potential of LCC Ca2�

currents are unable to evoke any sparks (57, 240, 390).Blockade of LCC gating (verapamil, nifedipine) or Ca2�

permeation (Cd2�) also inhibits spark production (58, 65,239). In contrast, spark activation in skeletal muscle iscontrolled by dual mechanisms, primarily depolarization-induced Ca2� release (DICR) and CICR. The former refersto the conformational coupling between the DHPR (volt-age sensor) and the RyR (release channel) (197). BothDICR and CICR trigger subsurface sparks in neurons (95).

It has long been known that stretching cardiacmuscle causes enhanced [Ca2�]i transients and contrac-

tion, a cellular basis for Starling’s law of cardiac func-tion (greater contraction is generated by greater pre-load of the heart). Stretch-induced Ca2� release (SICR)(Table 6) has recently been demonstrated at the level ofelementary releases. Ca2� spark production is propor-tionately enhanced by cell stretch up to 20%, and thiseffect is completely abolished by the nitric oxide syn-thase (NOS) inhibitor NG-nitro-L-arginine methyl ester(L-NAME) and is absent in cardiac myocytes from en-dothelial nitric oxide synthase (eNOS)-null mice (294).In urinary bladder smooth muscle myocytes, SICR inthe form of Ca2� sparks and propagating Ca2� wavescan be demonstrated in Ca2�-free extracellular solution(183). Similarly, Ca2� sparks independent of Ca2� entryare observed during integrin-mediated mechanotrans-duction in vascular smooth muscle cells (8).

X. SPARK MECHANISMS: COORDINATION

IN a CRU

A. Overview

Delineating the number of RyRs activated in a sparkis a matter of fundamental importance to sparkology.Depending on species and muscle type, a CRU in striatedmuscle contains a variable number of homotetramericRyRs, ranging from a few tens up to a few hundred of

TABLE 6.—Continued

Refractoriness ofCICR

A state in which CICR displays reducedresponsiveness to a Ca2� trigger.

See Refs. 48, 324. In a CRU, inactivation of RyR and depletion ofthe cistern connected to the CRU would both contribute to localrefractoriness. Recovery from this refractoriness underlies Ca2�

spark restitution. In DRG neurons there is no evidence ofrefractoriness of CICR mediated by RyR3 (284).

TABLE 7. How are Ca2� sparks triggered in heart?

With 100–300 RyR2s in the RyR2 cluster at the jSR, it is thought that the opening of any RyR2 homotetramer is as good as any other to initiatethe Ca2� spark. It is the event that leads to the first opening of a RyR2 in a CRU that constitutes the trigger of the spark, and after that, itappears to evolve autonomously, independent of the trigger. If the RyRs are tightly coupled and act in unison, the trigger is the event thatmakes the first multichannel unit to open simultaneously. In the extreme case, all it takes to trigger the spark is a single Ca2� in thesubspace that binds to a RyR in a CRU.

In a model of parsimony, the spark trigger mechanism is exclusively mediated by the aforementioned CICR mechanism. In this model, the vastspark-activating mechanisms we discussed in the text and Table 6, including SCICR, SOICR, SICR, and even DICR, can be viewed asmechanisms that “tune” the CICR, i.e., the probability of opening of the first RyR (or coupled RyR unit) by the subspace Ca2�. For instance,DICR reflects removal of tonic inhibition of the DHPRs on the RyRs, permitting the latter to respond to subspace Ca2�. For SCICR or SOICR,it can be considered as Ca2� acting from inside the lumen to alter the sensitivity of CICR on the cytosolic side.

It would be of interest to demonstrate whether any spark-activating mechanism is independent of CICR. An unequivocal proof of such amechanism could be difficult: ideally, spark activation should be demonstrated in the absence of Ca2� occupancy of the RyRs on thecytosolic side prior to activation of the first RyR. It is also noteworthy that not all RyRs in a CRU are at the same status with respect toligand (including Ca2�) binding, molecular partner association, and phosphorylation and other posttranslational modifications. Besides, thoseat the border of a CRU are not fully engaged in the RyR-RyR interaction at all four corners of the homotetramer. As a result, some RyRs maydisplay a higher propensity than their mates as the first RyR to be triggered in a spark.

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these SR Ca2� release channels. They are assembledinto a 2D paracrystalline array of round or elongatedgeometry (Fig. 8). The vast majority of RyRs are foundin such CRUs, the exquisite supramolecular assembly iscommon to all three RyR isoforms, and this is evolu-tionarily conserved from invertebrates to mammals(117, 130, 237). Teleologically, such RyR arrays mustexist for a purpose, performing biological functionsthat are impossible or less than optimal if RyRs exist asrogue channels. Reporting a CRU in action, Ca2� sparkscan best serve as an investigative tool to reveal Na-ture’s secrets hidden in this supramolecular Ca2� sig-naling nanomachine.

Deceptively simple, channel stoichiometry has per-vaded the field of sparkology ever since its inception.Because RyRs are intracellular channels, direct electro-physiological investigation of RyRs in intact cells hasthus far been impossible. There are many conceivablemeans by which the Ca2� release channels in a CRU

can interact. Each RyR2 homotetramer could gate in-dependently, interacting only through information inthe common Ca2� source (the SR lumen) or the com-mon Ca2� sink (the subspace). Because of the physicalconnectedness, these Ca2� release channels could alsooperate with a degree of cooperativity. There is a seriesof questions that, when answered, will influence ourthinking and constrain the models that can account forboth spark activation and spark termination. 1) Doesthe behavior of a CRU reflect the sum of the behavior ofindividual independent RyRs, or does it reflect novelgating mechanisms unseen in those acting solo? 2) Is afraction or are all RyRs in a CRU activated in a spark?Do these activated channels act in unison or not?3) Does [Ca2�]SR remain constant or does it declineduring the Ca2� spark? 4) Does [Ca2�]SR affect RyRgating? 5) Is there coupled gating of RyRs? 6) Is thereRyR inactivation or “adaptation” (152) (Table 6) in aspark?

FIG. 8. Organization of cardiac and skeletal ryanodine receptor (RyR) arrays. A: front and side views (inset) of RyR arrays in an invertebrate skeletalmuscle of cardiac-type EC coupling (i.e., via the Ca2�-induced Ca2� release mechanism). About a hundred RyRs (marked by arrows) are assembled intoa 2-dimensional lattice. Such assembly is thought to be representative of RyR arrays in cardiac myocytes. [From Loesser et al. (237). Courtsey of C.Franzini-Armstrong.] B: conceptual model showing a cardiac RyR array relative to LCCs in the plasma membrane. C and D: organization of RyR arraysin mammalian versus frog skeletal muscle. A couplon in frog skeletal muscle (C) consists of a central double row of RyR1 (blue squares), which arealternately coupled to DHPR tetrads (red circles, TT not shown), and a significant presence of RyR3/� (gray squares) in a parajunctional position (117).D shows the lack of a parajunctional RyR3/� array in a mammalian skeletal muscle couplon (129). [Modified from Ward and Lederer (394).]



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B. Key Observations

Seven key observations in cardiac cells guide thecurrent investigations and provide a context for pastfindings. 1) The Ispark is �3 pA and lasts for �10 ms (69,351, 390, 392). 2) The fractional Ca2� current through asingle RyR2 channel reconstituted in planar lipid bi-layer has been measured to be smaller than initiallyestimated and appears to be within the range of 0.5– 0.7pA under physiological ionic conditions (194). 3) Poly-morphism of Ca2� sparks can be demonstrated forevents from the same CRUs (331, 390). Nevertheless,the amount of Ca2� released and the duration of releaseare somewhat stereotypical. 4) Special conditions canlead to very long Ca2� embers at about half of the peakspark amplitude (69, 346, 420). 5) Substantial depletionof cisternal Ca2� develops during a spark (48). 6) Many(50 –300) RyRs are clustered to form each CRU as notedabove (130, 352) 7) Recent results suggest that theremay be substructure (quanta) in Ispark and the numberof quanta in a spark varies from event to event (390,392). In these studies, Wang et al. used sparklets pro-duced by a known LCC current as an optical “yardstick”and scaled the rising phase of a spark to infer the Ispark.The histogram of this calibrated Ispark suggests peaksevery 1.2 pA, as if sparks consist of Ca2� quanta of 1.2pA Ca2� flux (Table 1). However, little is known as towhether a quantum corresponds to a single RyR or agroup of tightly coupled RyRs. If such a spark substruc-ture is supported by future experiments, it will cer-tainly be an important basis for developing mechanisticinsights into the behavior of RyR2 CRUs.

C. CICR and Coupled Gating

Once an LCC or RyR opens, Ca2� floods into thesubspace and can thereby activate other RyRs. This CICRis central to all views of Ca2� spark triggering and acti-vation, and clearly involves a high-gain positive feedbackregulation. Based on the discussion above, that latency, is�10 ms at 0.3 pA Ca2� flux, and the power law of sparkactivation, we predict that latency,apparent is 0.9 or 0.06 msat 1 or 4 pA Ca2� flux, respectively. Thus we might expectthat CICR alone is sufficient to activate all RyRs in afraction of a millisecond. Thus, if not all RyRs in a CRUare activated in a spark, there must be an equally power-ful, yet-to-be-found inhibitory mechanism that inactivatesthe channels even before they can be activated.

The second mechanism invokes the notion that RyRsinteract via conformational coupling. In this manner, gat-ing of the RyRs in the CRU involves cooperativity amongthe RyRs, which is also known as “coupled gating” (249,251), since it was first identified in planar lipid bilayerexperiments with two RyRs that appeared to open and

close synchronously. How much does cooperative gatingcontribute to RyR activation in a spark? To a first approx-imation, not much. Since subspace CICR is already ex-pected to be powerful, an additional activation mecha-nism would be redundant. Nonetheless, one importantbenefit of cooperative RyR gating is to confer a steeperCa2� dependence for CRU activation compared with ac-tivation of rogue RyRs (347) (Fig. 9). CRUs can thus bemade stable at resting or low [Ca2�] but highly responsiveto subspace trigger [Ca2�]. Indeed, Stern and colleagues(367) showed that no published gating schemes of indi-vidual RyRs incorporated into EC coupling models canexplain the systolic and diastolic features of cardiac ECcoupling simultaneously; introducing cooperative gatingamong arrayed RyRs effectively cures the poor behaviorof the models. Such cooperative gating would provide acompelling rationale for packing RyRs into large arrays.

Albeit intuitively and theoretically appealing, littledirect evidence is available as to whether cooperativegating of RyRs occurs in a CRU, and if so, whether itinvolves subgroups or the entire CRU. If subgroups ofRyRs engage in cooperative gating, is the subgroupingstatic or dynamic? Answering these important yet difficultquestions calls for ingenious experimental ideas and in-novative techniques and promises a new level of under-standing of the operation of arrayed RyRs in intact cells.

FIG. 9. Rogue RyRs underlie sparkless release: a hypothesis. Plotsshow model predictions of steady-state Po as a function of cytosolic[Ca2�] for isolated (i.e., “rogue”) RyRs (solid line) and strongly coupledRyRs (solid circles and dashed line). In this model, the energetic cou-pling between RyRs represses the channel activity at low [Ca2�], causesa steep transition at a critical [Ca2�] level, and enhances the channelactivity at higher [Ca2�]. One implication of this is that rogue RyRs tendto be more easily activated by small increases in intracellular [Ca2�]above the resting level than clustered RyRs that gate cooperatively. Therelease from rogue RyRs in response to low-level trigger Ca2� thus mightmanifest as sparkless release. [Modified from Sobie et al. (347).]

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D. How Many RyRs Open During a Spark?

There is no simple answer. Reflecting on the range ofspark models proposed, we examine two schools ofthought, the “one and a few channels” model (16, 69, 141,321) and the “whole-CRU” model (30, 179, 346).

The first model suggests that only a tiny fraction ofRyRs in a CRU are activated to release Ca2� in a spark.The supporting evidence includes the average 4 pA flux,which is not far from that of a few RyRs; the plateau ofmodified cardiac sparks at �50% of the peak level, pre-sumably reflecting the sustained opening of a single RyR;and the quantal substructure in Ispark (if a quantum rep-resents a single or a small number of RyRs). The problemsare twofold, however. The first is a sort of teleologicalconsideration: Why on earth would only a small percent-age of RyRs be activated in EC coupling? Could it be asolution to the pressing need of maintaining stability atdiastole while enabling high-gain amplification at sys-tole? The second is: What prevents RyRs from activat-ing when exposed to high [Ca2�]subspace? This modelhas to invoke either a Ca2�-dependent inactivation thatis much stronger than previously thought (392), ornegative cooperativity among RyRs (an open RyR re-presses neighboring RyRs from activation), which is yetto be experimentally demonstrated. In this model, allRyRs in a CRU participate in the “to activate or toinactivate” computation, but only one or a few end upopen, and the others are inactivated.

The whole-CRU model, however, states that nearlyall RyRs in a CRU are activated by CICR and coupledgating during a spark. In this model, spark terminationdoes not invoke channel inactivation, but rather, a pow-erful luminal Ca2� desensitization of CICR upon cisternalCa2� depletion. The supporting evidence includes thelack of rapid and powerful channel inactivation in vitro,and on the demonstration of luminal Ca2� regulation ofCICR sensitivity (150, 151, 153, 328, 422; see also Ref.359). This model is also consistent with the somewhatstereotypical amount of Ca2� and release duration in aspark (392). Furthermore, it predicts that, as long as aCRU contains many RyRs, spark generation should berobust and independent of the number of RyRs in a CRU.For long-lasting sparks, the peak and plateau ratio nowdepends on the time-dependent cisternal Ca2� depletion.Some degree of polymorphism is expected from stochas-tic variation in CRU open duration. The problem of thetiny unitary current per RyR (�4 pA for 100 RyRs or�0.04 pA/RyR) could possibly be accounted for by adiminishing electrochemical potential of Ca2� {due to therise of [Ca2�]subspace (346, 350) and the fall of cisternal[Ca2�]SR (48)}. How such a model can accommodate theIspark substructure (392) remains to be determined.

XI. SPARK MECHANISMS: TERMINATION

CICR, in its simplest form, is expected to produceeverlasting local release of Ca2�. That Ispark lasts only5–10 ms is indicative of powerful termination mecha-nism(s) that turn off the elemental Ca2� signals, sharplyand promptly. So, what terminates a spark? To date, nosingle mechanism can adequately account for spark ter-mination, and more questions have been raised than an-swered. In this section, we discuss current concepts andexperimental evidence pertinent to spark termination.

A. Local Ca2� Depletion

Emptying the cisternal Ca2� store could, in principle,extinguish the spark. Significant cisternal depletion is alsoa requisite for deactivating or desensitizing RyRs via theSICR mechanism operating in reverse mode (Table 6)(150, 151, 153, 185, 276, 328). As discussed above, briefand substantial jSR Ca2� depletion does develop during acardiac spark, and evidence for this comes from the Ca2�

blink that mirrors the spark (48). However, store deple-tion cannot be the sole mechanism of spark terminationbecause of a simple “counterexample”: the rate of cister-nal refilling, measured as the recovery time of a blink, isabout six times faster than the rate of restitution of Ca2�

spark amplitude (48). Similar results are obtained whenthe restitution is measured by other means including theprobability of spark reactivation (349). Inferred from theirdata in skeletal muscle, Launikonis et al. (213) proposedthat CSQ, a high-capacity Ca2� buffering protein in thecisternal lumen, depolymerizes and reduces its Ca2�-bind-ing capacity upon store release. If this is applicable tocardiac myocytes, the slow recovery of spark amplitudecould reflect a slow restoration of the CSQ polymer-bound Ca2� pool. Taken together, these data suggest thatlocal store depletion may work in synergy with additionalinhibitory mechanisms for spark termination and restitu-tion.

B. Stochastic Attrition

“Stochastic attrition” refers to the inherent randomclosing of individual channels that can turn off an activatedchannel cluster (363) (Table 6). Albeit appealing for parsi-mony, stochastic attrition works only if a single or very fewRyR channels (or units of coupled gating) are activated in aspark (363). For stochastic attrition alone to terminate CICRin a CRU, the predicted release duration would increaseastronomically with increasing number and Po of indepen-dently gated channels in the spark. Furthermore, stochasticattrition does not impose any after-effect, in contrast to thelocal refractoriness of CICR that prevents a CRU from im-mediate reactivation (48, 324).



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C. Desensitization of CICR by Ca2�

Store Depletion

The sensitivity of the RyRs to be activated by cyto-solic Ca2� is augmented by increasing luminal Ca2� (67,150, 243, 346). When the local store depletes, not only dothe Ispark and [Ca2�]subspace diminish, but also the RyRsdeactivate or desensitize. That is, the SCICR can operatein reverse mode. Recent advances in Ca2� blinks supporta substantial local store depletion in a spark, a requisitefor this mechanism to work in intact cells. In a theoreticalmodel, Sobie et al. (346) have shown how luminal depen-dence of cooperative gating among RyRs can underlierobust Ca2� spark termination without Ca2�-dependentinactivation.

D. Coupled Gating as a Termination Mechanism

In contrast to the prevalent view on coupled gating asa CRU activation mechanism, we emphasize here that it ismore properly considered as a release stabilization andtermination mechanism. In other words, the RyR-RyRinteraction in a CRU may impart a powerful basal repres-sion of spontaneous release in resting cells and terminatethe release during a spark (347) or a global [Ca2�]i tran-sient (367). Putative RyR inhibition of neighboring RyRs(in a CRU) is analogous to the DHPR inhibition of RyR,which has been experimentally demonstrated in skeletalmuscle couplons (206, 443).

E. Channel Inactivation and Adaptation

RyR inactivation can be Ca2� dependent or “fateful”(296). Fateful inactivation refers to spontaneous transi-tion of an open channel to an inactivated state via aCa2�-independent mechanism. In a broad sense, the Ca2�

dependence of inactivation may involve binding of Ca2�

to inactivating sites, Ca2�-dependent phosphorylation ordephosphorylation of the channel per se, or of a molecu-lar partner in the macromolecular complex. The net effectis the loss of channel activity in the presence of sustainedor new stimuli.

1. Ca2�-dependent inactivation

Early demonstration of CICR by rapid application ofsolutions of defined [Ca2�] to mechanically skinned car-diac Purkinje fibers revealed a bell-shaped Ca2� depen-dence of force generation, with the peak at low micromo-lar [Ca2�]. At a given [Ca2�], the faster the applicationrate, the greater the CICR effect (114–116). From theseexperiments, Fabiato (116) postulated that Ca2�-depen-dent inactivation of high affinity but slow kinetics occursconcurrently with CICR. Nabauer and Morad (270) tested

Ca2�-dependent release inactivation in intact cardiacmyocytes. They showed that a preconditioning elevationof cytosolic [Ca2�] (from 90 to 110 nM for �500 ms) doesnot prevent ICa from triggering substantial release. Thisobservation has been widely interpreted as evidenceagainst a Ca2�-dependent inactivation in situ. This argu-ment, however, may be flawed. Their observation can bereadily explained if the Kd of inactivation is well abovethe conditioning [Ca2�] involved. Moreover, the condi-tioning protocol would upload Ca2� into the SR due to theelevation of steady-state [Ca2�]i and therefore mask theinactivating effect, if any. A similar argument was madeby Pizarro et al. (296) against similar proposals of inacti-vation by relatively low Ca2� in skeletal muscle (340). Inanother experiment, they showed that photolytic release ofCa2� delivered 50–100 ms after the onset of depolarizationduring the rise of contraction always potentiates cell con-traction. This observation is not inconsistent with Ca2�-dependent inactivation either, because the photolytic Ca2�

not only can directly contribute to cell contraction, but alsocan elicit CICR on its own. Insights gleaned from mathemat-ical modeling (350) show that [Ca2�]subspace rises to the 100�M range in a spark, suggesting that local Ca2�-dependentinactivation remains an appealing possibility, perhaps it issuch local inactivation that matters in terminating a spark(but see discussion elsewhere).

2. Planar lipid bilayer experiments

A reductionist approach to the spark mechanism is toinvestigate RyR single-channel behavior in planar lipidbilayers. However, it should be cautioned at the outsetthat many regulatory features, including interactions be-tween RyRs and their molecular partners (252), arestripped away in such experimental settings (Table 8).Direct single-channel measurement suggests that neitheractivation nor inactivation features of RyRs are simple.Inactivation, when it is observed, is too slow at physio-logically relevant [Ca2�] levels. Is inactivation of RyRinvoked? Yes. However, the inactivation and its charac-terization are complicated by varying experimental con-ditions and methods. There are multiple “kinds” of inac-tivation reported in the literature, and in this focusedpresentation, we divide the reports into two groups forthe sake of simplicity. There is a complex Ca2�-dependentslow inactivation called “adaptation” (66, 122, 152, 383)and a slow complex inactivation that we will call “other.”The “other” group is unified by the denial of adaptation asa valid inactivation mechanism (209, 210, 343). None ofthe “other” inactivation schemes is fast enough to enableRyRs to inactivate in a few milliseconds (165, 215), theapproximate rate needed to underlie spark termination.The rate of adaptation inactivation ranges from 150 ms toseconds, while the rate of the other forms of inactivationrange from more than a second to longer than 30 s (165,

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215). Clearly, as they stand, neither adaptation nor “other”inactivation is fast enough.

3. RyR channel adaptation

A complex kind of inactivation called “adaptation”(152, 383) (Table 6) is observed following a rapid increaseof cytosolic Ca2� by photolysis that produces an in-creased Po. Adaptation describes the decline in Po ofRyRs, and it differs from inactivation because an“adapted” channel retains responsiveness to subsequenthigher [Ca2�] steps (152) (Table 6). Conceptually, theadaptive behavior at the single-channel level can be ex-plained on the basis of complex time-dependent modalgating schemes (66). It should be cautioned that not everygroup can demonstrate the reactivation of the “adapted”channel by a second, higher [Ca2�] stimulus, particularlywhen purified RyRs are involved (see Refs. 122, 210 forreviews). There has also been discussion in the literatureabout the possibility that there is a complex time courseof [Ca2�]i following photolytic uncaging of Ca2�, and thiscomplex time course may contribute to adaptation (211,384). Adaptation, when found, is relatively slow (100 msto seconds) and thus kinetically inadequate to account forCa2� spark termination (ms to tens of ms), but couldcontribute to changes in RyR responsiveness over alonger time scale.

In crayfish skeletal muscle, where EC coupling ismediated by the CICR mechanism, Yasui et al. (431) dem-onstrated that depolarization to �30 mV in the presenceof FPL64176 elicits a transient Ca2� release that termi-nates despite continued ICa. Yet, additional Ca2� releasecan be triggered by tail ICa upon repolarization. UsingCa2� spike measurement, Sham et al. (324) found that thetail current produced on repolarization elicits Ca2� spikesthat most likely originate from RyRs unfired during depo-

larization, rather than from those in the adapted state.After a maximal activation, a multifold increase in ICa (byhyperpolarization to �120 mV) at 50 ms interval fails toevoke any additional release, indicating absolute refrac-toriness of CICR. These results argue in favor of inactiva-tion, rather than adaptation, as the specific mechanism ofrelease refractoriness (and spark termination). However,the high degree of intracellular buffering that significantlyslows jSR refilling may complicate the interpretation.

F. Refractoriness of Local CICR

Following a global Ca2� transient or wave, triggeredCa2� release becomes refractory. Refractoriness of globalCICR is exemplified by annihilation of two Ca2� waves inhead-on collision and suppression of depolarization-acti-vated [Ca2�]i transients in the wake of a Ca2� wave. It hasbeen thought that the mechanisms of refractoriness mayhave a lot in common with those that terminate the re-lease in the first place.

Refractoriness of local CICR has been convincinglydemonstrated by different groups. An early approach byCheng et al. (67) examined restitution of AP-elicited Ca2�

release in the wake of a Ca2� wave and suggested thatrestitution may be as fast as �150 ms. Tanaka et al. (371)showed that, immediately following a spontaneous spark,there is oftentimes a void at the sparking site when aglobal [Ca2�]i transient is elicited by an AP. This approachsuggested that restitution of Ca2� release takes at least 25ms. DelPrincipe et al. (96) showed that there is littlechange in the triggered release of Ca2� produced by longpulses of two-photon uncaging of Ca2�, but restitutionover periods less than the minimum time between pulses(200 ms) would have been missed. Sham et al. (324) usedthe Ca2� spike method to investigate Ca2� release trig-

TABLE 8. Generic differences between RyRs gating in situ and in vitro

In a Spark In Planar Lipid Bilayers

Organization Assembled into large-scale, 2D paracrystalline arrays. In vertebrateskeletal muscles, forming conformational coupling with DHPRsin the plasma membrane.

Individual channels act solo in most cases. Coupledgating can be demonstrated only to a limitedextent (two or a few RyRs).

Molecular partners Exist as macromolecular complexes and decorated with a largepanel of molecular partners on the cytosolic side, in the ER/SRmembrane, and inside the ER/SR lumen. Native ionic conditionsand native lipid constituents.

Likely be stripped of most of the molecularpartners, particularly after channel purification.Even if retained, the stoichiometry and spatialrelationship are likely distorted. Experimentallydesigned ionic conditions; artificial lipidconstituents.

Dynamic Ca2� signals Responding to brief (0.1–10 ms), high level (10–500 �M) subspace�Ca2�� produced by triggering LCC or RyR release flux. Dynamicluminal �Ca2�� changes occur as in Ca2� blinks.

Steady-state behavior at different �Ca2�� isexamined in most studies. Adaptational responsesto step increases of �Ca2�� and brief Ca2� pulseshave been demonstrated with monovalent cationsor divalent cations as the charge carrier. ConstantCa2� on the luminal side.

Gating and regulatorymechanisms

Sparks are the collective behavior of the supramolecular signalingsystem, with local positive and negative feedback and feed-through loops. See Table 6 for spark mechanisms.

Single-channel behavior reflects the intrinsicproperty of the channel or in the presence of afew tightly associated elements.



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gered by depolarization and by repolarizing ICa tail cur-rents. Use-dependent inhibition of triggered Ca2� releasewas observed, but the kinetics of the findings are difficultto interpret because of the significant level of cytosolicCa2� buffer.

Terentyev et al. (375) and Sobie et al. (349) usedImperatoxin A in permeabilized cells and low levels ofryanodine in intact myocytes to investigate Ca2� sparkrestitution. These agents induce repetitive Ca2� sparksand can thus be used to examine spark restitution. Sobieet al. (349) reported that the amplitude of Ca2� sparksrecovers with a time constant of 91 ms, while the trigger-ing probability recovers with a time constant of �80 ms.Using a loose patch-clamp method, Brochet et al. (48)found that the sparklet-spark coupling fidelity and theamplitude of the succeeding spark decrease substantiallyfollowing spark activation. The recovery of local refrac-toriness appears to be much faster (spark amplitude res-toration with a half-time of �150 ms) than the restitutionof global CICR (half-time 600 ms); the latter is rate-limitedby the refilling of the SR Ca2� store. Taken together, thesefindings suggest that local refractoriness does developand the Ca2� blink measurement suggests that the impor-tant region of depletion is the cistern. However, cisternalrefilling alone cannot fully explain the kinetics of restitu-tion, necessitating the involvement of other mechanismswith slower kinetics.

G. RyR Gating In Vivo Versus In Vitro

At the heart of the uncertainty and controversy re-garding the mechanisms of spark activation, coordina-tion, and termination is the paucity of information on RyRgating in situ. It has been increasingly appreciated thatcaution must be exercised in translating results from theplanar lipid bilayer to intact cells, and vice versa. Genericdifferences between RyRs in a spark and those in planarlipid bilayers are summarized in Table 8. In the first place,gating of RyRs in a spark is constrained by the 2D arrayassembly to which they belong. The array formation is aproperty intrinsic to the RyR, as purified RyRs can formlarge-scale arrays in solution or on the surface of posi-tively charged lipid membrane (432, 433). In a skeletalmuscle couplon, DHPRs also physically constrain the gat-ing of RyRs in the junctional CRU. Second, the channelbehavior in a spark may be modified by a large number ofmolecular partners in the macromolecular complex onthe cytosolic side (e.g., calmodulin, calstabin, sorcin, ki-nases, phosphatases) (47, 222, 250, 252, 259), in the lipidmembrane (e.g., triadin, junctin), and inside the lumen(e.g., CSQ). Most of these are dislodged during vesiclepreparation and particularly by channel purification priorto reconstitution. Third, the steady-state behavior studiedin vitro does not serve as a good predictor of transient,

nonequilibrium responses of the channel, for there is nounique or simple relationship between kinetic and steady-state responses. Last but not least, many positive- andnegative-feedback loops (e.g., local CICR, desensitizationby luminal Ca2� depletion, Ca2�-dependent phosphoryla-tion and dephosphorylation) could be involved in shapingthe sparks. Not all these gating and regulatory mecha-nisms can be readily reproduced in reductionist ap-proaches in vitro. With these differences kept in perspec-tive, we can judiciously integrate the findings in cell-freesystems with the spark measurement in intact cells. Bothtypes of approaches are indispensable in defining howRyR behavior is linked to Ca2� sparks.

XII. Ca2� SPARKS IN HEART DISEASE

Diseases of the heart may affect Ca2� sparks andtheir properties. These changes reflect the alterations inmany regulatory features of the RyR2 macromolecularcomplex, such as altered RyR2 itself and its associatedproteins, spatial organization of the RyR2 clusters, inter-cluster distance, sensitivity of RyR2 to [Ca2�]i, the SRCa2� content, and Ca2� “leak” from the SR, as well as APduration and shape. Recent findings suggest that diversediseases (e.g., diabetes, muscular dystrophy) with cardio-vascular consequences may also contribute to cardiacCa2� signaling dysfunction. With respect to these newfindings and current exploration of treatments involvingstem cells and novel small molecular therapeutic agents,much work remains to be done to characterize the linksbetween diseases and altered behavior of Ca2� sparks andSR Ca2� release. Importantly, inventive mathematicalmodeling may provide new understanding of Ca2� sparksand Ca2� signaling in heart and lay the foundation forfuture experiments and analysis.

A. Arrhythmic Disease

An early clue that Ca2�-activated current contributesto arrhythmogenesis came from studies of current oscil-lations in heart that were attributed to SR Ca2� overload(191, 219). Follow-up investigations revealed small cur-rent fluctuations that were linked to mechanical and[Ca2�]i “noise” (60, 189, 365, 412). The arrhythmogenicmembrane current, identified as the “transient inwardcurrent” (ITI) appeared to arise from a synchronization ofCa2�-dependent current fluctuations and what would turnout to be a propagating wave of elevated [Ca2�]i (20). ITI

can activate sufficient inward current to produce arrhyth-mias in two ways. The first is to augment early afterde-polarizations (EADs) due to the interactions of ICa andrepolarizing K� currents (219); the second is to producede novo delayed afterdepolarizations (DADs) (118, 119,

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219, 388). Both EADs and DADs can produce aberrantelectrical activity and extrasystoles (arrhythmias).

This classic view of Ca2�-dependent arrhythmogen-esis was initially clarified by the discovery of Ca2� sparkswhen it was shown that Ca2� waves are initiated by a“triggering” Ca2� spark in the condition of Ca2� overload(69). Ca2� overload is the condition that occurs whenthere is an elevated amount of Ca2� in the SR. The highSR Ca2� content makes the SR unstable (190). That theCa2� wave is linked to ITI at the cellular level becameclear in multiple studies (20, 87, 193, 242, 355, 408). Thisdid not address the question, however, whether or notthese events are linked to arrhythmogenesis in the intactheart. Early work did suggest that the underlying cellularevents can be “scaled up” to account for arrhythmogenic-ity in intact cardiac tissue (408). Some of the complexitiesof these connections are discussed below.

How the waves of elevated Ca2� are propagated inheart cells is central to the understanding of the role ofCa2� sparks in arrhythmogenesis. Early examinationshowed three relevant features of Ca2� waves. 1) Asnoted above, they appear to be initiated by Ca2� sparks.2) The propagation depends on sequential propagatingactivation of Ca2� sparks by the locally elevated Ca2� dueto the earlier Ca2� sparks (67). 3) A common featureidentified is the increased sensitivity of the CICR processthat enables Ca2� sparks that would not normally activateneighboring Ca2� sparks to do so (67, 178, 180, 408).

Ca2� overload itself as an RyR2 “sensitizer” plays animportant role in producing Ca2� instability in heart un-der many conditions. While it was first described follow-ing blockade of the Na� pump by cardiotonic steroids (82,83, 109, 191, 219), it also plays a key role in the develop-ment of Ca2� instability in a variety of conditions. Forexample, increased SR Ca2� load has been put forward asa central factor in increased RyR2 sensitivity following�-adrenergic stimulation (225), although phosphorylationof RyR2 may also influence the process (see below). Ca2�

overload is an important contributor to the generation ofCa2� alternans (103, 108, 138) and in many channelopa-thies (see below).

B. Heart Failure

There are many distinct insults that presage heart fail-ure (HF) and for much of this discussion, we mean left-sidedHF. In this late-stage condition, the left ventricle is unable topump enough blood to meet the demands of the tissue. HFis produced in reaction to acute pump failure or sustainedexcess afterload or both, and pulmonary edema often devel-ops during HF. The acute injury that leads to HF is severebut not lethal as in an acute myocardial infarction (MI).Following that insult, the unaffected tissue must compen-sate for the infarcted tissues, and this leads to “remodeling”

of the healthy cells. In a similar manner, the stress of hyper-tension is not necessarily acutely associated with HF, butthe HF develops with sustained hypertension and conse-quent cardiac remodeling. In these contexts, the remodelingis the response of healthy tissue to sustained excess workand is associated with the onset of Ca2� signaling dysfunc-tions. While it may provide some adaptive benefit ini-tially, the remodeling becomes maladaptive when itcontinues in the face of sustained demand. There aresix clear changes that appear to occur with this mal-adaptive remodeling: 1) reduction in the SR Ca2� pump,SERCA2a (93); 2) increase in NCX level (342, 421); 3)prolonged APs (see below); 4) altered TTs (361); 5) thedevelopment of dyssynchronous Ca2� release (233); and6) increased SR Ca2� leak (see below).

C. Remodeling of the Ca2� Signaling System

1. CRU organization

The spatial organization of the CRUs may change indisease and affect the likelihood that a Ca2� spark at oneCRU will trigger a Ca2� spark at a neighboring CRU. Asnoted above, there is �1 CRU/fl (�m3) in rat ventricularmyocytes separated by 0.65 �m in the plane of the Z-diskand by �0.16 �m from the centroid of the Z-disk (352)(Fig. 10). The couplons (made up of between 120 and 260RyRs) in one Z-disk are separated from the couplons inthe next Z-disk by the sarcomere spacing (�1.8 �m inhuman and rat). Despite the asymmetry of couplon spac-ing, Ca2� waves, when observed, are near-spherical. Can-nell and colleagues (352) suggest that this may comeabout because of the asymmetry of longitudinal (faster)versus transverse (slower) diffusion (68). In this regard,anisotropy of Ca2� diffusion appears to dictate the dis-parity of inter-CRU distance in differenct directions. Suchpossibilities are being investigated by several groups.

2. SERCA2a and NCX

The reduction in the expression of SERCA2a leads to areduction in the amount of Ca2� in the SR, and this alonewould reduce the [Ca2�]i transient. The increase in NCXexpression would tend to reduce the SR Ca2� still furtherbut would predispose to the generation of a larger ITI for agiven increase in [Ca2�]i during a Ca2� wave (298). Thedepolarization-produced ITI is enhanced further by the lowerlevel of repolarizing K� current that is also found in HF (298,312). Exactly how changes in SERCA2a and NCX are pro-duced and the molecular signaling involved remain un-known, but there are some clues about Ca2� and nuclearfactor of activated T-cell (NFAT) signaling pathway contrib-uting to the changes in K� current (312). The major questionis how does the elevation of [Ca2�]i come about? What rolesare played by Ca2� sparks? How does this come about in acellular environment that has, in principle, reduced total Ca2�?



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3. Dyssynchrony of Ca2� sparks

Song et al. (360) first proposed that synchrony ofCa2� sparks is an important determinant for physiologicalmodulation of cardiac EC coupling. A key observationwas made by Litwin, Zhang and Bridge in the context ofEC coupling in HF (233). They observed that in cells froman MI model of HF, Ca2� sparks do not behave normallyduring the AP (233). Although depolarization of the HFmyocyte activates a somewhat diminished ICa, the sur-prise comes from the timing of the Ca2� sparks. The Ca2�

sparks are not synchronized with the depolarization, and“late” Ca2� sparks are present so these Ca2� sparks are“dyssynchronized” with respect to the depolarization. Iso-proterenol treatment of the afflicted HF ventricular myo-cytes leads to reduction of the dyssynchronization (233).The many actions of isoproterenol on the heart cell makethis observation useful, but it does not point to a morespecific cause.

Kamp and co-workers (9), using tachycardia-inducedHF, suggested that Ca2� signaling dysfunction arises fromdepletion of the TTs in HF heart cells. While this seems aless subtle cause of dysfunction than dyssynchronizationof Ca2� sparks may have required, the concept of TT“disease” is appealing in principle. Sipido and colleagues(241) examined pig ventricular myocytes in culture thathad lost TTs and compared their findings to human ven-tricular myocytes from individuals in HF, supporting thehypothesis that TT loss is linked to dyssynchronous Ca2�

sparks. Using spontaneously hypertensive rats (SHR) inHF, Song et al. (358) showed that the TTs (that have apreponderance of LCCs) appear to dedifferentiate andbecome increasingly disorganized in SHR ventricularmyocytes in HF while the CRUs and the RyR2 clustersassociated with the SR remain largely in place (Fig. 11).There is thus increasing separation between the trigger

(ICa) for Ca2� sparks and the RyR2 release channels.Recent work of Kamp and co-workers (254) supportsthese conclusions by simultaneously imaging TTs andCa2� sparks in HF.

4. SR Ca2� leak

RyR2-mediated Ca2� efflux from the SR is thought todepend on two components: triggered Ca2� release and“leak” Ca2� loss. While triggered SR Ca2� release under-lies the normal [Ca2�]i transient and involves Ca2�

sparks, the leak loss of Ca2� from the SR occurs duringdiastole. In addition to spontaneous Ca2� spark contribu-tion to the leak, there is also the probability of RyR2sopening as rogue RyR2s (1 to about 5 RyRs in a cluster) sothat there is no visible Ca2� spark (346, 414). If RyR2sbehave cooperatively when they are in a cluster (249),then the [Ca2�]i dependence of Po is much steeper (Fig.9). This means that with large clusters, as are seen in theCRU, cooperativity may be significant, and with that highdegree of cooperativity, there is a reduced likelihood thatthe cluster is triggered at low [Ca2�]i. The rogue RyR2swith no or small degrees of cooperativity may be morereadily activated than CRUs by low [Ca2�]i. In the sim-plest circumstance, when there is an increase in SR Ca2�

leak without other changes, there is a decrease in SR Ca2�

content and hence a decrease in the tendency to produceCa2� sparks and Ca2� waves (29). Thus such an increasein Ca2� leak tends to be antiarrhythmic. Recent work,however, has suggested a “paradox of Ca2� signaling” inthe diseased heart: there is an increased leak, and this SRCa2� leak is associated with arrhythmias (18, 220–222,377, 385, 398, 399).

It is, however, a challenge to square the relation-ships between and among the signals with the require-ments of SR pump-leak balance, the tendency of one

FIG. 10. Constellation of Ca2� release units (CRUs) in cardiac myocytes. A: CRU distribution in a transverse section of a human ventricularmyocyte. Each circle represents a CRU detected after deblurring of confocal RyR-immunofluorescence images. B: color-coded map of the areasserved by each CRU in a Z-disk of a human ventricular myocyte. Scale bar: 2 �m. [See Soeller et al. (352).]

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Ca2� spark to trigger a wave and of Ca2� sparks tosustain the propagation of the wave. This is a clearexample where mathematical modeling of Ca2� sparksand Ca2� waves within a single cell is needed to informour thinking on how the arrhythmias arise, what rolemay be played by Ca2� sparks during the initiation, howCa2� sparks may contribute to sustained activation,and how leak and “invisible” SR Ca2� release maycontribute to Ca2� wave propagation. Some of theseconsiderations provide a context for future modelingand experimentation.

5. AP duration and arrhythmias

Changes in AP duration and shape occur in diverseacquired and genetic diseases (78, 275). These changescan affect Ca2� sparks and Ca2� waves by changing SRCa2� load and diastolic [Ca2�]i through voltage-depen-dent action on Ca2� entry and the NCX Ca2� effluxacross the surface membranes. In addition, the changesin AP profile alter the triggering of Ca2� sparks (46, 59,146, 313, 345). Thus the tendency for the ventricular APto be longer in HF and other diseases, often due to lessrepolarizing K� current (188, 207, 216, 260, 269, 406),tends to promote both Ca2� loading and Ca2� sparks.

When EADs develop due to prolonged APs, they areattributed to interactions between the repolarizing K�

currents and the depolarizing Ca2� currents (92, 161, 281,282) or possibly larger than normal inward Na� currents

(1, 78, 123). The EADs can, however, be significantlyaffected by Ca2� sparks and Ca2� waves via ITI (20, 73, 89,219), the latter making them more readily reach thresholdto trigger an extrasystole. DADs, in contrast, depend pri-marily on Ca2� sparks and Ca2� waves to activate ITI.Importantly, however, the amount of NCX that is ex-pressed and is functional in a cell, as well as the amountof K� current present, appear to play an important role indetermining how large the voltage excursion is when aCa2� wave actives ITI (298).

D. Genetic and Acquired Channel Dysfunction

Ion channel dysfunction may have a profound effecton Ca2� sparks and Ca2� waves through gain- or loss-of-function mutations or by other means. For channels thatpass Ca2� or Na�, the ion flux may lead to Ca2� overloaddirectly or indirectly (via NCX). Loss of function muta-tions or drugs that block K� channels lead to prolongedAPs. Channel trafficking problems can lead to mistarget-ing of ion channels so that they end up in the wrongmembrane microdomain or get stuck at some point in theprocessing. For example, K� channel dysfunction may beassociated with ion channel trafficking errors (10). Whenthe abundance of functional K� channels is decreased(due to any cause including trafficking errors), APslengthen. Prolonged APs increase SR Ca2� load byfavoring Ca2� entry and decreasing Ca2� extrusion and

FIG. 11. Intermolecular relationships between LCCs and RyRs in normal and diseased heart cells. A: TT disorganization in ventricular myocytesfrom failing SHR heart (SHR/HF) and age-matched control (WKY). B: immunostaining of RyRs (top) and LCCs (bottom) in SHR/HF and WKYventricular myocytes. Note the chaotic distribution of LCCs in the diseased heart cell. [A and B from Song et al. (358), copyright 2006 NationalAcademy of Sciences, USA.] C: diagram of TT-SR structure.



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may lead to EADs and Ca2�-dependent arrhythmogen-esis as noted above. Excess Na� entry through theexpression of late Na� channels or poorly inactivatingINa (132) can affect EADs but can also increase theintracellular Na� concentration and hence [Ca2�]. Itwas appreciated over 30 years ago that the intracellularNa� level has a significant affect on Ca2� in the SR andhence on the cellular vulnerability to Ca2� instability(110, 190, 268), and this has been seen in diseasedrodent heart cells (3, 101). The elevation of intracellularNa� leads to an increase in Ca2� sparks, Ca2� waves,and ITI and thus acts to produce arrhythmias through aCa2� overload mechanism.

RyR2 dysfunction can also develop, and this pro-duces the “leaky” RyR2 that has been widely presented inliterature. Specifically mutations in RyR2 (248) increaseits Po at constant [Ca2�]i and make ventricular myocytesunusually vulnerable to triggered or spontaneous activity.The molecular mechanisms by which this may happenremain uncertain because of the dual-dependency ofRyR2 Po on Ca2�; it is sensitive to both [Ca2�]i and[Ca2�]SR as noted above. Since the rapid triggering of aCRU, is affected by the opening of an apposing DHPR, themore parsimonious explanation is that local [Ca2�]i “trig-gers” the CRU, while [Ca2�]SR “tunes” the sensitivity oftriggering (Table 7).

Just as RyR2 mutations may increase the Po of RyR2sby increasing their sensitivity to [Ca2�]i, mutations inproteins associated with the RyR2 can do the same. Mu-tations in the cardiac isoform of CSQ, CSQ2, have alsobeen recently reported to increase RyR2 Po and sensitivityto [Ca2�]i and cause Ca2� instability, Ca2� sparks andwaves, and arrhythmias (76, 105, 151, 154, 155, 201, 234,326, 353, 374). The other associated proteins (junctin andtriadin), when they are mutated or expressed at abnormallevels, may also bring about altered sensitivity of RyR2 to[Ca2�]i and contribute to arrhythmogenesis (17, 131, 135,143, 151, 155, 196, 373, 376).

E. Cardiac Ca2� Signaling in Diverse Diseases

Complex diseases may affect heart function and, inmany cases, the links between the primary disease andthe myocyte remain obscure. Diabetes is associated withCa2� signaling dysfunction including abnormal Ca2�

sparks and Ca2� release (75, 94, 329, 341, 429, 430). Dys-trophin mutations account for Becker and Duchennemuscular dystrophy, the two most common muscular dys-trophies. While both are primarily diseases of skeletalmuscle, each has severe cardiac consequences; amongother things in mouse models there appear to be abnor-mal Ca2� signaling in heart (187) and altered mitochon-drial function. In each of these cases, the molecularcauses of the cardiac signaling dysfunction are beinginvestigated.

XIII. SPARKS AND EMBERS

IN SKELETAL MUSCLES

A. Overview

In a vertebrate skeletal muscle couplon, type 1 RyRs ina CRU form a double row assembly; tetrads of DHPRs inthe plasma membrane overlay and form conformationalcouplings with RyR homotetramers, but only at alternateRyRs (129) (Fig. 8). In this “double checkerboard” ar-rangement, the RyRs unconnected to DHPRs are appar-ently in direct contact with those that are engaged withDHPRs. In the skeletal muscle of frog and other nonmam-malian species, there are also parajunctional type 3 RyRstripes along the jSR that run parallel on either side of thedouble RyR1 swath (Fig. 8) (117). The presence of suchRyR3 tetramers or the lack of them confers distinctivephenotypes on the respective muscles: sparks in amphib-ians and embers in mammals (197, 300, 381) (Fig. 12).Investigation of the lack of sparks, the presence of em-bers, and the reappearance of “ghost sparks” (394) (Table1) in mammalian skeletal muscle as well as the sparkmechanisms in amphibians has led to the appreciation oftwo new mechanisms controlling the skeletal muscleCa2� release from store. The first is related to an inhibi-tory role of DHPR on RyR1 and the second, the require-ment of parajunctional RyR3 in supporting depolariza-tion-evoked sparks. Next, we first discuss the activation,coordination, and termination of skeletal muscle sparkswith reference to their counterparts in heart and thensparkless release, embers, and the conditions for reap-pearance of sparks in mammalian skeletal muscles.

B. Sparks in Amphibians

1. Activation mechanisms

First suggested on the basis of noise analysis, spon-taneous Ca2� sparks and those induced by depolarizationor RyR ligands have been extensively characterized inamphibian skeletal fibers. In permeabilized or cut-endpreparations, skeletal muscle sparks are wider (2–4 �m)but briefer (3–5 ms rise-time, 15 ms FDHM) than cardiacsparks (197). They are localized to regions where cou-plons or TT-CRU junctions are expected (197, 381). Re-ducing [Mg2�] (from 2 to 0.1 mM) profoundly enhancesthe rate of spark production (197, 205, 442), suggesting aninhibitory role of intracellular Mg2� at physiological con-centrations. Interestingly, caffeine not only increases thespark frequency, but also makes sparks wider, which wasinterpreted as recruiting more channels into the spark(16, 142). Baylor et al. (16) carefully examined importantmorphometric differences between sparks from the intactfiber preparation (with indicator loaded by microinjec-

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tion) and the cut-open preparations: sparks in intact fibersare briefer (FDHM � 6 vs. 9–15 ms), narrower (FWHM �1.0 vs. 2.0 �m), and 3- to 10-fold smaller by signal mass.These differences suggest that diffusible cellular constit-uents and exact ionic conditions are important in deter-mining the CRU gating, the Ispark, and the appearance ofsparks.

Dual mechanisms, DICR and CICR, control depolar-ization-evoked release, which does not require any Ca2�

entry and is largely made up of sparks (141, 197, 199) (Fig.12). Spark activation at around threshold voltages dis-plays an e-fold increase of rate occurrence every 4.1 mV ofdepolarization, similar to the voltage-dependent activa-tion of DHPR, the voltage sensor. By brief repolarizationto reprime inactivated voltage sensors in depolarized frogfibers, Klein et al. (199) resolved sparks over the full rangeof skeletal muscle fiber depolarization (from �60 to �40mV) and demonstrated autonomy of Ca2� spark forma-tion irrespective of voltage and latency.

2. Coordination mechanisms

As is the case for cardiac sparks, the estimates forthe number of RyRs activated in a skeletal muscle sparkcover a wide range. In one school of thought, the “one ora few channel model” explains spark production, becausefeatures of intact muscle sparks, including spark width,

can be reproduced by model simulation with a pointsource Ispark of 1–3 pA (63). The time course of Ca2�

sparks in amphibian skeletal muscle has been meticu-lously examined with video-rate (63 �s/line) confocal mi-croscopy (206). The fluorescence time course is system-atically well fit by an empirical equation consisting of anexponential rising phase followed by an exponential fall-ing phase. This feature is consistent with an immediateand full activation of the release flux at the start of aspark, constant throughout the rising phase, and suddenand complete cessation of the release at the peak of thespark. Since spark formation involves low-pass filteringcomponents (Ca2� buffering, indicator kinetics, diffu-sion), the optical measurement cannot resolve brief clo-sures during an ongoing spark. Hence, such a squarewavelike flux can be generated by sustained or burstingopening of either a single channel or a channel group(321).

In the competing school of thought, amphibian skel-etal muscle sparks arise from up to a 30 pA release flux(based on model extraction from the brightest sparks),requiring the activation of as many as 60 RyRs (306). Theestimated release flux underlying an ember induced byImperatoxin A is merely 1/8 to 1/20 of the estimated peakIspark. The center of an ember may be off-set from thecenter of its precedent spark by a fraction of a micron,

FIG. 12. Sparks and embers in amphibian andmammalian skeletal muscle fibers, respectively. A, left:x-y scan image from a frog semitendinosus muscle atrest, voltage-clamped in a 2-Vaseline gap chamber, andequilibrated with an internal solution containing fluo 3.Right: line scan (along the line in blue on the left); adepolarizing pulse was applied as indicated. The imageis pseudo-colored for visibility. B: line scan image of arat extensor digitorum longus (EDL) fiber, voltage-clamped in a 2-Vaseline gap clamp, and similarly equil-ibrated with a fluo 3-containing internal solution. Thevoltage protocol starts from a depolarized situationand includes an interval of 2.8 s at �90 mV. This allowsfor partial recovery of the voltage-inactivated voltagesensors, which results in a reduced density of activerelease channels and improved resolution of individualembers. [Modified from published and unpublishedwork of E. Rios, J. Zhou, N. Shirokova, and L.Csernoch.]



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indicative of an extended source of Ispark. Moreover, fast2D imaging visualizes a wavelike evolution of a spark: thewavefront propagates over 1–2 �m at a speed of 100 �m/s,as if the spark is generated by CICR along the direction ofan elongated skeletal CRU (366, 441). Caffeine increasesthe propensity and propagation speed of the wavelikesparks. Importantly, the appearance of wavelike sparksalso suggests that RyRs in a skeletal muscle CRU do notgate in unison, arguing against a tight CICR or coupledgating under the experimental conditions.

An amphibian couplon consists of the DHPR voltagesensor, the junctional RyR1 array, and parajunctionalRyR3 arrays on the same cistern (Fig. 8). During a depo-larization-evoked spark, junctional RyR1 may serve as themaster to control the activation as well as termination ofthe slave RyR3 arrays (206, 300). The mechanism bywhich these RyR1 and RyR3 arrays communicate witheach other presumably involves CICR on the cytosolicside and, perhaps, Ca2�-dependent regulation in thelumen.

3. Termination mechanisms

Skeletal muscle sparks are more abruptly terminatedthan their cardiac counterparts and exhibit an averagerise time of �5 ms. As in cardiac cells, use-dependentrefractoriness of spark production has been demon-strated by several groups (197, 198, 214). Only �7% cis-ternal depletion (213) or �10% global store depletion(286) is associated with a spark or a muscle twitch, re-spectively, in amphibian skeletal muscle, suggesting alimited contribution of cisternal depletion to spark termi-nation. Compared with RyR2, RyR1 is readily inactivatedby a Ca2�-dependent mechanism, and its Po displays abell-shaped Ca2� dependence with profound inactivationat millimolar [Ca2�] (256, 423). However, RyR3 in theparajunctional arrays is known to be resistant to inacti-vation even in the presence of 10 mM [Ca2�] (238).

A novel termination mechanism, “termination byvoltage sensor deactivation,” has been demonstrated fordepolarization-evoked sparks (Table 6). Lacampagneet al. (206) have shown that DHPR deactivation on repo-larization curtails an ongoing spark, violating spark au-tonomy. They suggested that continued DHPR activationis essential to sustain the evolution of a spark in the firstfew milliseconds of spark generation. Beyond this timeperiod, the release inactivation mechanism would termi-nate the spark independently of DHPR. This finding is ofgreat importance for several reasons. First, it shows that,in contrast to the cardiac counterpart, a skeletal spark is,to some extent, still under the control of the DHPR duringits evolution; the autonomy of skeletal muscle sparks isnot absolute. Second, DHPR deactivation terminatesCICR, if any CICR occurs between junctional (RyR1) andparajunctional channels (RyR3) and within the junctional

array. If we envisage the immediate effect of DHPR deac-tivation is the cessation of Ca2� flux from the DHPR-coupled RyR1, it can be inferred that CICR in a sparkoperates in a low-gain regime (G � 1) and is unable tosupport regenerative release after removal of the triggerCa2�. Third, DHPR may repress RyR1 activity at rest,serving to inhibit SR Ca2� leakage at rest. This idea is nowsupported by more direct evidence from mammalian mus-cle (443). Hence, DHPR in skeletal muscle functions notonly as the voltage sensor and the Ca2� entry pathway,but also the release terminator and the SR leak repressor.

C. Embers and Sparks in Mammals

1. Embers and ghost sparks

The quest for elementary release events in adultmammalian species first met with a surprise: the almostabsolute absence of spontaneous sparks in intact quies-cent adult fibers (332) and, during depolarization, the lackof evoked Ca2� sparks (335). Subsequent studies undercertain experimental conditions (e.g., inclusion of SO4

2� inthe internal solution) (91, 440) revealed that depolariza-tion-evoked release consists of lone embers: long-dura-tion, low-amplitude (�F/F0 � 0.2), low flux (�0.5 pA)release events (Fig. 12). These lone embers are narrower(1.3 �m) than sparks and lack the initial peaks seen inspark-trailing embers in amphibian skeletal muscle in thepresence of RyR modulators. A true irony, however, isthat mammalian skeletal muscle does generate spark-featured releases under certain circumstances: robustspark production after saponin permeabilization of thesurface membrane or in the presence of SO4

2� in theinternal solution in cut-end preparations (440), or locallyin membrane-permeabilized ends (91). Moreover, typicalsparks and long-lasting Ca2� “bursts” (401) (Table 1) ap-pear in intact rat skeletal muscle fibers upon exposure tohypertonic solution, or returning from hypotonic swell-ing, or after strenuous excerise of the animal (393). Ahigher propensity of Ca2� spark activity has also beenassociated with aging skeletal muscle (258, 400, 402).Thus the same Ca2� release machinery is capable ofproducing no sparks (in quiescent fibers), sparks, or an-other type of elemental release event, in stark contrast tothe amphibian and cardiac counterparts. These resultssuggest that mammalian skeletal muscle does contain thespark-generating machinery, but it is normally repressedto manifest sparkless release; experimental and patho-physiological conditions can unmask these “ghost sparks”(394).

2. Inhibitory role of conformational coupling between

DHPRs and RyRs

While the traditional view is that DHPRs serve solelyas the voltage sensor to their RyR1 partners in physical

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contact, the current understanding is that DHPRs, whennot activated, prevent their RyR1 partners from activa-tion. The first clue was from the observation that voltagesensor deactivation abbreviates an amphibian spark asdiscussed above. More recently, it has been shown thatrepolarization truncates ongoing embers in rat skeletalmuscle (440). These findings indicate that DHPR activa-tion and deactivation tightly control the commencementand termination of CRU release. The observation of loneembers in mammalian skeletal fibers provides a hint. Ifthe embers each arise from a single RyR, their merepresence implies little or no CICR (or coupled gating)among RyR1 channels in the same junctional array. Thereappearance of sparks after chemical permeabilization,exercise, or osmotic shock from otherwise sparkless re-lease is generally consistent with this idea, as if subtlemembrane deformation disrupts DHPR inhibition ofCICR. In immature (85, 86, 336), differentiating (443), ordedifferentiating (50) mouse skeletal muscle fibers, depo-larization evokes a sparkless type of release at spatiallysegregated sites, whereas discrete sparks occur at loca-tions devoid of direct EC coupling (and hence DHPRinhibition). Additional evidence comes from investigationof Ca2� release in myotubes from dysgenic mice (mdg)that lack DHPRs but contain both RyR1 and RyR3 iso-forms. By comparing mdg with the wild type, Zhou et al.(443) have shown that DHPRs reduce the rate of sponta-neous opening of the release channels and their suscep-tibility to activation by high Ca2� and 1 mM caffeine. ThusDICR, DHPR-dependent spark repression, and spark ter-mination by voltage sensor deactivation appear to bedifferent manifestations of the same underlying mecha-nism (see Ref. 394 for a review). Physiologically, directvoltage control of both release activation and terminationaffords tight control of Ca2� signaling by brief APs (ofmillisecond duration) and, in quiescence, strong inhibi-tion of background SR Ca2� leak in skeletal muscles. Adisruption of such an inhibitory mechanism or reappear-ance of ghost sparks is associated with skeletal muscleaging and dystrophy (393, 400).

3. Role of the RyR3 isoform

The striking species difference between amphibiansand mammals in spark production has been linked to theirrespective CRU organization (300, 335). Only in the am-phibians are there parajunctional sets of RyRs (presum-ably RyR3) not directly controlled by the DHPRs (117);CICR activation of accessory RyR3 is thought to accountfor the appearance of spontaneous sparks when junc-tional RyR1 channels are normally inhibited by DHPRs.This hypothesis is supported by the observation that tet-racaine eliminates the depolarization-evoked sparks butspares the smaller, diffuse, and sparkless release in am-phibian skeletal muscle fibers (335). In an elegant study,

Pouvreau et al. (300) used electrotransfection of cDNA toexpress either RyR3 or RyR1 in mouse skeletal muscleand showed that exogenous expression of RyR3, but notRyR1, enables spontaneous sparks and macrosparks atrest and produces abundant sparks upon depolarization.It has been concluded that depolarization-evoked sparksin skeletal muscle require a voltage sensor, a masterjunctional RyR1 channel that provides the trigger Ca2�,and a slave parajunctional RyR3 cohort (300). Conversely,the lack of embers in amphibians and at RyR3-expressingmammalian couplons suggests that the parajunctionalRyR3 cohort may enslave the junctional RyR1 for releasetermination, via perhaps depletion of the common cister-nal Ca2� pool.

XIV. SMOOTH MUSCLE SPARKS

As in striated muscle, Ca2� sparks are present in awide variety of smooth muscle myocytes including thosefrom artery (133, 262, 274), portal vein (144, 263), urinarybladder (84, 163, 164, 183, 266), ureter (42, 52), airway(203, 235, 339), and gastrointestinal tract (145, 405). Theyoccur spontaneously and can be evoked by depolariza-tion, either directly by CICR or indirectly by SCICR due toSR loading by Ca2� influx. Instead of an extensive reviewof spark mechanisms, here we focus on physiologicalfunctions consequential to Ca2� spark activation insmooth muscles. In particular, we illustrate how the highlocal [Ca2�] produced by sparks activates plasma mem-brane Ca2�-sensitive channels or electrogenic transport-ers to impart their physiological actions.

A. Activation of Ca2�-Sensitive Channels

by Subsurface Sparks

SR elements in close apposition (�20 nm) to theinner side of the plasma membrane have been observed indifferent types of smooth muscle myocytes (102, 223)with RyRs within 100 nm of the cell membrane (139, 223).A subsurface Ca2� spark, which measures 2–3 �m indiameter and occupies �1% of the cell volume, can affect�1% of the cell membrane (293), on which reside theCa2�-sensitive channels, including big-conductance Ca2�-activated K� (BKCa) and Ca2�-activated Cl� channels(ClCa), and electrogenic Ca2� transporters such as theNCX.

Spontaneous outward currents (STOCs) mediated byBKCa channels were first described by Benham andBolton (19) and were thought to arise from sudden dis-charges of the subsurface SR Ca2� store. In a classicwork, Nelson et al. (274) showed that subsurface ryano-dine-sensitive sparks activate iberiotoxin- and TEA-sensi-tive STOCs in arterial smooth muscle. Simultaneous re-cording of STOCs and sparks suggests that virtually every



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spark activates a STOC (293), indicating a near unityfidelity for spark-to-STOC coupling (� �1). In rat cerebralarterial myocytes, electrophysiological measurement ofsingle-channel and whole cell BKCa channel activity yieldsan estimate of 3,000 BKCa channels/myocyte or �2 chan-nels/�m2, and an average STOC amplitude of 148 pA andunitary BKCa current of �8.5 pA, at �40 pA and symmet-ric 140 mM K� concentration (292). From this, it is esti-mated that �18 BKCa channels open simultaneously at thepeak of a STOC. Furthermore, by determining the Ca2�

dependence of BKCa channels in excised patches (Kd � 19�M, Hill coefficient � 2.9 at �40 mV), the lowest estimatefor local [Ca2�] to trigger BKCa channels would be �4 �M(assuming 100% channels clustered over the spark) andthe highest [Ca2�]i estimate �30 �M (assuming uniformdistribution or 1% of channels affected by the spark)(292), similar to the [Ca2�] expected to be found in thesubspace of a cardiac couplon (350). Spark-to-STOC cou-pling appears to be extremely robust. Loading the myo-cytes with a fast Ca2� chelator, BAPTA, causes the dis-appearance of Ca2� sparks, but the STOCs remain un-changed (292), suggesting that the fast Ca2� chelator isunable to break the coupling, and the underlying Ca2�

signals that mediate the coupling must be too local to bedetected by current optical methods. In addition to theLCC-to-RyR coupling, RyR-to-BKCa coupling provides an-other good example of high, local [Ca2�] being sensed bya local target.

Such stringent local control of Ca2� signaling wouldpredict that BKCa activity can discriminate Ca2� signalsfrom different sources and, conversely, sparks can differ-entially affect different Ca2�-sensitive channels, depend-ing on relative localization between the source and thetarget. Indeed, it has been shown that, in urinary bladder

myocytes, blockade of RyR release inhibits iberiotoxin-and TEA-sensitive BKCa currents without affecting theCa2�-activated small-conductance K� channel (SKCa) cur-rents that are apamin sensitive. This suggests that SKCa

channels are somewhat excluded from the microdomainsof RyR CRUs. In contrast, Ca2� entry through voltage-dependent Ca2� channels (VDCCs) activates both BKCa

and SKCa channels, but with distinctive kinetics that sug-gest VDCCs are more distant from BKCa than SKCa chan-nels (164).

In addition to STOCs, sparks can activate ClCa chan-nels to produce transient inward currents (STICs) (Fig. 13)(166). When BKCa and ClCa channels coexist, spontaneoustransient outward then inward currents (STOICs) are re-corded (Fig. 12) (446). These STOCs and STICs are ex-pected to modulate Vm and excitability, with STOCs beinghyperpolarizing and inhibitory and STICs being depolar-izing and excitatory. In addition, the increase in mem-brane conductance (Gm) negatively influences membraneexcitability (see below). The spark-STOC and spark-STICcouplings, akin to sparklet-spark coupling, provide a real-time demonstration of intermolecular signaling events inintact cells.

B. Ca2� Sparks Relax Smooth Muscle

An increase in [Ca2�]i in arterial smooth muscle myo-cytes is expected to initiate muscle contraction, as is thecase in striated muscles. Counterintuitively, Ca2� sparksrelax smooth muscle. Nelson et al. (274) have shown thatthe underlying mechanism involves, sequentially, subsur-face Ca2� sparks, STOCs, membrane hyperpolarization,resulting shutoff of LCC Ca2� entry, and a loss of intra-

FIG. 13. Spark modulation of membranecurrents. Depending on membrane potential,three sparks from the same site (A: surfaceplots; B: time courses) in a tracheal smoothmuscle myocyte evoked a BKCa current(STOC), a combined BKCa and ClCa current(STOIC), and a ClCa current (STIC) (C) at 0,�50, and �80 mV, respectively (446).

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cellular Ca2� (Fig. 14). Specifically, the direct contribu-tion of sparks to global [Ca2�]i is almost inconsequential:a spark would raise the global [Ca2�] by �2 nM if its Ca2�

flux is uniformly distributed in the cytosol. The rate ofoccurrence of spontaneous sparks is only �1 Hz next tothe cell membrane, and thus the time-averaged change inglobal [Ca2�]i is even smaller. On the other hand, thespark-activated STOCs would cause a time-averaged hy-perpolarizing current of the order of 10 pA and a �10 mVhyperpolarization assuming a few G input resistance ofthe cell, leading to reduction of VDCC-mediated Ca2�

entry. Myocyte relaxation and vasodilatation ensue as aresult of the net reduction in [Ca2�]i (Fig. 14). That sparkscause arterial myocyte relaxation vividly exemplifies howthe same Ca2� messenger fulfils opposing physiologicalfunctions in a given cell.

A diverse group of vasodilators, including �-adrener-gic agonists through cAMP-dependent kinases and nitricoxide through protein kinase G, act on the spark-STOCaxis in regulating smooth muscle tone (195, 253, 302).They elicit parallel severalfold increases in the frequencyof sparks and STOCs. Defective spark-STOC coupling hasbeen linked to dysregulation of relaxation and hyperten-sion that may progress into heart failure (4). Amberg et al.(6) have shown that the BKCa channel displays bluntedsensitivity to sparks, due to altered expression of the�-subunit of the channel in the spontaneously hyperten-sive rat. Decreased coupling of sparks to BKCa in �-sub-unit knockout mice is associated with elevated mean

blood pressure and left ventricular hypertrophy, suggest-ing a role of spark-STOC coupling in blood pressure reg-ulation in intact animals (45, 297). In acute hemorrhagicshock, however, the BKCa channel sensitivity to micromo-lar [Ca2�] is enhanced, increasing the size of STOCs andcontributing to vasodilation and hypotensive conditionsas well as a blunted vasoconstrictive response to �-adren-ergic stimulation (439). Furthermore, it has been shownthat loss of BKCa, and hence spark-STOC coupling, leadsto penile erectile dysfunction (404).

C. Spark Modulation of Membrane Excitability

While arterial smooth muscle tone is regulated byvarying Vm, APs are generated in smooth muscles thatundergo phasic contraction. In this type of smooth mus-cle, oscillatory spark production may serve as a “Ca2�

clock” (208, 391) (Table 3) that is coupled to the electro-physiological clock made up of pacemaker ionic currents,and collectively, they set the rhythm of the firing of APs.For instance, phasic spark production has been shown toregulate the AP refractory period for the peristalsis ofureter smooth muscle, and is associated with an excep-tionally long refractory period (10–100 s) (42, 52). Ca2�

entry via LCC during an AP loads up the SR and increasesthe spark activity, perhaps via the SCICR mechanism.Spark activity gradually fades away in �70 s as the SRgradually depletes. Blockade of spark activation or BKCa

FIG. 14. How do sparks relax arterial smooth muscle and regulate vascular tone? A: subsurface Ca2� sparks promote membrane hyperpolar-ization through the activation of BKCa and STOCs in arterial smooth muscle myocytes. Hyperpolarization tends to shut off Ca2� influx via thevoltage-operated channels (VOC), resulting in a net decrease of intracellular Ca2� and relaxation of the arterial smooth muscle. B: in intact vessels,increases in intravascular pressure lead to membrane depolarization, activation of voltage-dependent Ca2� channels, and increased arterial tone.The spark-STOC coupling thus acts as a negative-feedback mechanism to limit pressure-induced arterial tone. [Modified from Nelson et al. (274).]



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channel activity causes premature firing of the next AP.Intriguingly, the spark modulation of membrane excitabil-ity occurs with no persistent hyperpolarization (52), incontrast to arterial smooth muscle tone regulation. Pos-sible explanations for the lack of significant hyperpolar-ization include 1) the resting Vm is closer to the reversalpotential of K� currents in phasic than tonic smoothmuscles, limiting the amplitude of STOCs; 2) the mem-brane input resistance might be lower (higher conduc-tance) such that STOCs are less effective in hyperpolar-izing the cell; and 3) there might be Ca2�-activated inwardcurrents, such as STICs, that nullify the outward currents.

Despite the lack of a direct Vm-hyperpolarizing effect,BKCa channel activation nonetheless increases in Gm andthus makes the membrane difficult to depolarize (i.e.,smaller voltage excursion in response to a given current),conferring the refractoriness. If this interpretation is cor-rect, the spark modulation of ureter smooth muscle re-fractoriness employs a novel “Vm-clamping” rather than aVm-hyperpolarizing mechanism. In summary, the firing ofan AP sets the Ca2� clock consisting of the SR, the RyR,and the BKCa channel modules; the Ca2� clock then slowsthe ticking of the electrophysiological clock until it itselftimes out; the electrophysiological clock then catches upwith the pace and sets off the next AP. Then, the nextcycle begins (Table 3).

XV. NEURAL Ca2� SPARKS

A. Characteristics and Mechanisms

An elaborate ER system is found in the cell body ofmany types of neurons, extending into the shanks ofdendrites and axons and perhaps to dendritic spines andpresynaptic terminals (23). The possibility of the exis-tence of Ca2� sparks as the elemental release events inneurons was first suggested on the basis of excessive“noise” or “outlier” bright pixels in neuronal Ca2� imagesin the absence of discernible local events (257). Mean-while, Koizumi et al. (202) demonstrated elementary Ca2�

release events arising from both RyRs and IP3Rs in nervegrowth factor-differentiated PC12 cells and cultured hip-pocampal neurons. These neuronal sparks exhibit 2-foldgreater spatial width and at least 20-fold longer durationthan a typical cardiac spark. Later, Llano et al. (236)visualized RyR-mediated spontaneous Ca2� release atpresynaptic terminals, where individual events last for afew seconds and spread 5–10 �m along the processes. Itis unknown whether these are neuronal sparks from sin-gle CRUs or complex supraspark events involving multi-ple CRUs. More recently, neuronal sparks termed “syntil-las” (95) (Table 1) have been characterized in hypotha-lamic neural terminals: these can be activated via theDICR mechanism. Another unequivocal demonstration of

neuronal Ca2� sparks is in mammalian DRG sensory neu-rons (Fig. 15), where sparks from RyR3 on the subsurfacecisternae are activated by physiological Ca2� influxes orthe RyR sensitizers caffeine and DMPX (283, 284). Highlylocalized (�0.66 �m width) 10-s-long ryanodine-sensitive“Ca2� glows” (427) (Table 1), as well as typical Ca2�

sparks, have also been observed in superior cervical gan-glion neurons in primary culture.

Compared with prototypical sparks in muscles, DRGsparks are characterized by a halved amplitude (�F/F0 �0.3), similar spatial extension, but prolonged release du-ration (�40 ms) (284). The small amplitude combinedwith the prolonged duration predicts a much smaller Ispark

in DRG neurons than muscle cells. In contrast to thehighly regulated release duration in muscles, the releaseduration of DRG sparks displays a monoexponential dis-tribution. Another distinctive feature is that long-lastingDRG sparks usually rise to their plateaus without initialovershoot. Collectively, the small flux, long and exponen-tially distributed release duration, and the waveform oflong-lasting events are all consistent with the possibilitythat DRG sparks are from a single or a few tightly coupledRyRs.

B. Properties of CICR in DRG Neurons

Virtually all CRUs in DRG cells manifest repetitivespark activity (283), indicating a lack of refractoriness.Though unexpected, this is consistent with the planarlipid bilayer observation that RyR3 from DRGs displaysno Ca2�-dependent inactivation even with 10 mM [Ca2�]on the cytosolic side. Yet, it was noted that the DRG CICRsystem is unable to support spontaneous propagatingCa2� waves under various experimental conditions.Quantification of cellular [Ca2�]i transients revealed thatCICR in DRG neurons (283) appears to operate in alow-gain, linear regime (G � 0.54). In this regard, Stern(363) showed mathematically that a CICR system with G

�1 is intrinsically stable in spite of positive feedback.These results illustrate that CICR in DRG neurons oper-ates in a regime of unconditional stability. Through alow-gain CICR system, DRG neurons can still generatelarge intracellular [Ca2�]i transients because the surfaceCa2� current density (156 pA/pF at �10 mV) is exception-ally high compared with that in cardiac cells (Table 9).The striking differences as well as the similarities be-tween neural and cardiac CICR (Table 9) are instructiveas to how the same signaling mechanism can be adaptiveand plastic in different physiological contexts.

C. Possible Role of Neuronal Sparks

The role of sparks is less well understood in neuronsthan muscles. The presence of extensive subsurface cis-

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ternae (SSC) implies that, among other possibilities, neu-ral sparks are instrumental in mediating bidirectionalcommunications between the plasma membrane and theER. The forward mode of communication, i.e., spark ac-tivation by both CICR and DICR mechanisms has beendemonstrated by independent groups. In the retrogradesignaling mode, spark modulation of membrane excitabil-ity, analogous to that in smooth muscle cells and cardiacpacemaker cells, represents an appealing possibility thatawaits future investigation (Fig. 15).

Ca2�-dependent vesicle secretion of transmitters andpeptides is widely involved in neural functions. Mostneurons release their neurotransmitters from terminalsat the synapse in a Ca2�-dependent manner. However,small DRG neurons (15–25 �m in diameter, C type) arecapable of Ca2�-dependent somatic exocytosis of,among others, the pain-related peptides, calcitoningene-related peptide and substance P (12, 170, 438).Emerging evidence suggests that somatic vesicle re-lease might be common to central nervous system neu-

rons (169). In this regard, Ca2� sparks from SSCs couldelevate local [Ca2�] to trigger exocytosis. Since neuralsecretion is a superlinear function of [Ca2�] (77),sparks can also modulate secretion through synergismwith depolarization-induced Ca2� influx. Both possibilitiesare supported by data from simultaneous measurement ofsubsurface Ca2� sparks and membrane capacitance (Cm)increases (as an index of vesicular secretion). Ouyang et al.(283) found that caffeine- or DMPX-induced Ca2� sparksincrease Cm, independently of membrane depolarization andexternal Ca2�. Inhibition of spark production by ryanodinesuppresses Cm changes and abolishes endotoxin-inducedsecretion of pain-related neuropeptides. By signal averaging,a tiny Cm change associated with individual sparks has beendiscerned, and the spark-secretion coupling fidelity in DRGneurons is �1 vesicle/10 sparks (� � 0.1). At present, itremains an open question whether spark-secretion couplingis generally involved in mediating synaptic release (see Ref.140 for a review of the possible role of local Ca2� release inthe regulation of axon pathfinding).

FIG. 15. Subsurface sparks and their possible roles in neurons. A: immunostaining shows subsurface localization of RyR3 puncta in a DRGsensory neuron from rat. “N” marks the nucleus. B: subsurface localization of sparks. C: possible functions of subsurface sparks in neurons.RyR/IP3R sparks from the ER cisterns, which can be activated by Ca2� entry through the voltage-operated Ca2� channels (VOCs), could modulateexocytosis and membrane excitability by activating BCa, ClCa channels, and NCX currents in the plasma membrane (PM). [Data from Ouyang et al.(284).]



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XVI. IP3R Ca2� PUFFS AND BLIPS

The cell logic is more complex for IP3R than forRyR-mediated Ca2� signaling, because the former re-quires both IP3 and Ca2� as coagonists (21, 125). A usefulview point is that IP3 serves as a physiological ligand totune the Ca2�-dependent inhibition of the IP3R releasechannel (246). IP3Rs are ubiquitously distributed and arethe common effector of many GPCRs and tyrosine kinasereceptors that are linked to the activation of phospho-lipases and the production of the second messengers IP3

and DAG from PIP2. It is generally believed that IP3Rs alsoform clusters or CRUs (289, 338), but direct ultrastruc-tural evidence is yet to be seen.

Ca2� puffs and mixed IP3R-RyR sparks have beenreported in many types of cells, including Xenopus

oocytes (287a, 289), HeLa cells (40, 156), oligodendro-cytes (156), neonatal cardiac myocytes (244, 299), andadult atrial cells (448), where they fulfill cell-specific func-tions. The Xenopus oocyte, which contains exclusivelytype 1 IP3R, has been a model system for investigatingIP3R signaling. Immunocytochemical data visualize adense distribution of IP3R to a subsurface band �5 �mthick, with the outermost layer separated by �6 �m fromthe surface membrane (54). A distinctive feature of IP3RCRU operation is that it can generate both puffs andsmaller release events (“blips”) (Table 1) in response tovarying IP3-uncaging light intensity (53, 310), i.e., grada-tion of Ca2� release at the level of single CRUs. Wespeculate that the concentration of IP3 determines theeffective size of a CRU, i.e., the number of IP3-occupiedchannels for CICR. A fully developed puff exhibits a meanintensity of 1.5 (F/F0 of Oregon Green 488 BAPTA-1) and3.5 the brightest, a long duration (100–600 ms), and alarge spatial spread (up to 6 �m). The properties of puffs

can be explained by the synchronous opening of 25–35IP3Rs, each carrying a Ca2� current of �0.4 pA, with thechannels distributed through a cluster 300–800 nm indiameter (310). As for the puff termination mechanism, itis well-documented that Ca2� exerts a biphasic action onsingle IP3R Po. In type 1 IP3Rs from a Xenopus oocyte,Ca2�-dependent channel activation occurs in an IP3-inde-pendent manner, with a half-activation concentration of210 nM. Ca2�-dependent inhibition, however, is tuned byIP3, with the half inhibition [Ca2�] varying from 160 nM to42 �M as IP3 concentration decreases from 180 �M to 10nM (246). Annihilation of colliding Ca2� waves indicatesthat the cytoplasm as an excitable medium becomes re-fractory in the wake of recent excitation (218). Because ofthe refractoriness and other nonlinear behavior of CICR,complex spatial and temporal patterns emerge, elegantlymanifested as spiral Ca2� waves (218). In Xenopus oo-cytes, the ClCa current displays a steep IP3 concentrationdependence. Due to the �10 �m spatial separation, thesurface membrane ClCa channels can hardly be affectedby rogue puffs but can be effectively activated by coordi-nated puff activation in the form of local or global Ca2�

waves traversing over the subsurface IP3R band (289).An intriguing feature of IP3R signaling, discovered in

the early 1990s, is the so-called “quantal release” (120,264, 380): cells respond to an IP3 signal with a [Ca2�]i

transient that decays almost completely to the restingCa2� level; however, a subsequent greater IP3 signal canstill elicit yet another [Ca2�]i transient of similar ampli-tude. By spike measurements, quantal release has re-cently been demonstrated at the single CRU level (55),indicating that it is a property intrinsic to the CRU. Atpresent, it is unknown whether the quantal release featurerequires channel clustering or is a feature of single-chan-nel adaptation (see discussion of RyR adaptation). Lumi-

TABLE 9. Properties of neuronal and cardiac CICR

In Ventricular Myocytes In DRG Sensory Neurons

Mechanism and molecular players CICR mediated by RyR2 CICR mediated by RyR3Trigger signal ICa (approximately �10 pA/pF at �10 mV) ICa at high density (�156 pA/pF at �10 mV)Elemental events Ca2� sparks from discrete CRUs Ca2� sparks from discrete CRUsArchitecture of control Subsurface and medullar CRUs are both under local

control because of the transverse tubule systemA hybrid architecture: subsurface CRUs under local

control, and medullary CRUs forming a common-pool system.

Peak Ca2� level Bell-shaped voltage dependence Bell-shaped voltage dependence�F/F0 approximately 3–4 (fluo 4 signal) at �10 mV �F/F0 approximately 3-4 (fluo 4 signal) at �10 mV

Refractoriness Strong use-dependent refractoriness Frequent bursting activity suggests littlerefractoriness

Capacity of store Ca2� reserve Substantial local store depletion during sparks Ca2� store can support all CRUs to fire �15 roundsLinearity Nonlinear power function of the trigger Ca2�

(power � 2)Linear function of the trigger Ca2�

Gain High-gain regime (G approximately 10–70) Low-gain regime (G � 0.54)Monotonic decreasing function of voltage Voltage independent

Stability Stable under normal Ca2� load Unconditionally stableInstability manifested as spontaneous Ca2� waves

under “Ca2� overload” conditionsUnable to support spontaneous Ca2� waves

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nal Ca2� regulation of IP3R sensitivity to cytosolic Ca2�

and IP3 has also been suggested as a candidate mecha-nism underlying the quantal Ca2� release.

All three types of IP3Rs are coexpressed, with type 2IP3R being predominant in developing heart (14, 229, 267).In mammalian adult ventricular myocytes, IP3Rs are ex-pressed at a low level and are thought to be concentratedat the perinuclear region and are found at the Z-line.These IP3Rs may be inconsequential to cardiac EC cou-pling but may play an important role in transcriptionalregulation (13, 419), evidenced by IP3R-mediated perinu-clear sparks and waves (244). However, there is no con-vincing demonstration of intranuclear IP3R-mediatedCa2� signals. In atrial cells, IP3Rs comingle with RyRs tomediate subsurface Ca2� sparks and are thus implicatedin cardiac arrhythmogenesis (448). The expression ofIP3Rs is generally elevated in various heart conditions(137, 424), suggesting possible roles in cardiac functionaland structural remodeling.

XVII. SPARKLESS RELEASE

Despite the fact that sparks are ubiquitous in differ-ent tissues and cells, a sparkless type of release is asso-ciated with ectopic expression of cardiac or skeletal RyRsin Chinese hamster ovary cells (32, 33), whereas expres-sion of RyR1 or RyR3 in myotubes null for native RyRsproduces sparks (395, 396). Even in heart cells whereCa2� sparks predominate, sparkless release has also beendocumented. NCX in the reverse mode (extruding intra-cellular Na� in exchange for extracellular Ca2�) triggersrelease that is far more uniform than expected if sparkswere the elementary release units. Furthermore, Niggliand Lipp initially reported sparkless releases upon Ca2�

steps produced by ultraviolet photolysis in cardiac myo-cytes (232), but later revised their conclusion by showingvariable local release events triggered by two-photon fo-cal photorelease of caged Ca2� (230). In oocytes, a small,spatially uniform Ca2� signal immediately upon photo-lytic production of IP3, termed “pacemaker Ca2�,” hasbeen shown to precede the occurrence of IP3R blips andpuffs (247). Sparkless and subspark release events havealso been systematically investigated in adult mammalianskeletal muscles (see above).

Sparkless release indicates that the underlying ele-mentary release events involve quantities of Ca2� that arebeyond the detection limit in these cases, suggesting thatCa2� sparks do not account for the totality of ER/SRrelease under all circumstances. The coexistence ofsparks and sparkless release in heart cells is difficult toreconcile if both are based on the same CRUs. To this end,Sobie et al. (347) speculated that there are solitary, yet-to-be-visualized RyRs scattered over the ER/SR, or “rogueRyRs,” either in transit toward the junctions or by biolog-

ical design. The rogue RyRs might be more sensitive tolow levels of Ca2� but less sensitive to high levels of Ca2�

than arrayed RyRs, the latter displaying a steeper, highlycooperative Ca2�-activation curve (Fig. 9). Besides gatingkinetics, the connected store capacity and molecular part-ners on both cytosolic and luminal sides may also differfor rogue and CRU RyRs. Release via rogue channels,rather than CRUs, might also account for the sparklessrelease in the ectopic expression system and for the pace-maker Ca2� signal of IP3-induced sparks.

XVIII. NEW INSIGHTS INTO Ca2� SIGNALING

A. Mechanisms Underlying Excitation-Ca2�

Release Coupling

Distinctive mechanisms are used in different types ofcells to trigger store Ca2� release upon membrane depo-larization. As discussed above, direct coupling in skeletalmuscle as well as in some types of neurons involvesconformational interaction between the voltage sensorand the release channel, independent of Ca2� entry. TightCICR coupling in cardiac as well as invertebrate skeletalmuscles operates in the nanoscopic subspace of cou-plons. In contrast, many types of smooth muscle (e.g.,urinary bladder and arterial smooth muscles) display an“indirect or loose coupling” between membrane excita-tion and Ca2� release (84, 113, 164). Features of theindirect or loose intermolecular communication betweenLCCs and RyRs include the following: 1) triggered Ca2�

sparks are often observed long after LCC closure; 2) CICRis a function of total LCC Ca2� influx, rather than iLCC;and 3) CICR is sensitive to cytosolic exogenous Ca2�

buffers. It may also involve an SR Ca2� reuptake andSCICR component. In this regard, Essin et al. (113) re-cently demonstrated that disruption of LCC expression inarterial smooth muscle cells leads to fewer STOCs ofreduced amplitude, accompanied by reduced cytosolicand SR Ca2�. This effect is fully reversed after restorationof the global and SR Ca2� levels.

Both direct and tight couplings can provide the speedand stability for the mobilization of stored Ca2�. Oneparticular advantage of direct coupling is perhaps relatedto its ability to inhibit CICR when the muscle is notactivated (443), which is of obvious advantage in bioen-ergetics, noise reduction, and prevention of false firing.Furthermore, direct coupling may also be a requisite forsharp termination of the release flux when an AP lastsmerely a few milliseconds (206). In heart, however, theAP is of much longer duration (hundreds of milliseconds),so tight CICR coupling appears to suit the purpose betterto release Ca2� in systole and then completely terminateit in diastole, the latter being crucial to cardiac relaxationfor refilling. In the situation of smooth muscles with tonic



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depolarization and very long AP duration, signal sustain-ability might dictate the choice of possible mechanismsthat couple Ca2� influx to store release. CICR via global,rather than, local [Ca2�]i transients would greatly prolongthe signal duration. If this is coupled with store Ca2�

uptake and SCICR, the Ca2� release signal would lasteven longer. In this way, distinctive coupling mechanismsmay fulfill cell type-specific functions.

B. Ca2� Sparks and the CICR Paradox

CICR as the ubiquitous control mechanism of ER/SRCa2� release was once thought to be paradoxical. WithCa2� as both the input and the output, CICR represents apositive feedback, ensuring the needed sensitivity, speed,and amplification of Ca2� signals. However, uncontrolledCICR is expected to operate in an all-or-none fashion,unless the gain of CICR amplification is sufficiently low(G � 1) (279, 363). In contrast, cardiac EC coupling as ahigh-gain CICR system is characterized by a smoothlygraded response to the trigger ICa in terms of amplitudeand duration.

To solve this CICR paradox, early experimental andtheoretical work invoked the “local control theory” ofCICR (Fig. 16) (279, 363). This theory consists of fourpostulates, each of which has now been independentlyvalidated by findings stemming from sparkology.

1) RyRs in situ are essentially insensitive to global[Ca2�]. The rate of occurrence of spontaneous Ca2�

sparks shows an opening rate of merely 10�4 s�1 for RyRat the resting [Ca2�] of �100 nM (69). Likewise, experi-

ments with Ca2� influx by Na�/Ca2� exchange or suddenuniform increase in cytosolic [Ca2�] produced by photol-ysis have also shown that CICR is a low-gain amplificationsystem in response to physiological levels of global [Ca2�](0.1–1 �M) (228, 279, 323, 325). 2) Yet, RyRs are effec-tively activated by local [Ca2�]. The extremely high effi-cacy of CICR in a couplon is exemplified by the ability ofa single LCC sparklet to trigger local release at a micro-scopic gain of �600 (see above). The peak rate of pro-duction of triggered sparks is estimated to be on the orderof 106 sparks per cell per second (58). Hence, cardiacCICR manifests both high-gain amplification (within thecouplons) and low-gain behavior (when triggered byglobal, low-level [Ca2�]). The nonlinear power functionfor Ca2�-dependent spark activation (315) provides thecritical mechanism to discriminate between the subspaceand the ambient [Ca2�] such that CRUs are quiet at dias-tole and highly active at systole. 3) Once activated, theRyR-mediated Ca2� signal remains local. The discrete-ness of Ca2� sparks suggests that individual CRUs op-erate independently of each other, and global CICR is,in essence, the digital summation of individuallyevoked sparks in space and time. The spatial locality ofCICR is safeguarded by many factors acting in synergy,including the physical separation of CRUs, the sheer[Ca2�] gradients, the strong Ca2� buffering of the cy-tosol, and the insensitivity of RyRs to low-level Ca2�. 4)A robust mechanism terminates CICR within a CRU.The brevity of Ca2� sparks ensures the temporal local-ity of CICR, but the exact nature of the spark termina-tion mechanism remains elusive.

FIG. 16. Sparks and the CICR para-dox. A: in a common pool model, high-gainCICR is expected to be explosive, operat-ing in an all-or-none fashion. B: the sparkmodel (or local control model) is able tosupport high-gain CICR within a CRU,while CICR between and among adjacentCRUs is abrogated. C: when the Ca2� sig-naling system is perturbed and made un-stable (e.g., with increased SR Ca2� load),spatially regenerative activation of CRUsmanifests as propagating Ca2� waves.

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In a nutshell, the discrete architecture of couplon,the extremely high-gain amplification within a couplon,the extremely low-gain amplification between CRUs,and the prompt termination of couplon activation pro-vide the basis for local control of CICR and account forthe paradoxical Ca2� phenotypes in the heart. Theseinsights have general implications in understanding thecontrol of Ca2� signaling in different types of cells.

C. Cellular Architecture of Ca2� Signaling

The discovery of Ca2� sparks and other elementaryCa2� signals has revealed new insights into the organiza-tional principles and cellular logic of the Ca2� signalingsystem. In the prespark era, it was thought that Ca2� risesand falls uniformly in the cytosol, a continuous and ho-mogeneous excitable common pool. Coding and decodingthe biological information are achieved by Ca2� concentra-tion and time as two analog variables. It is now understoodthat nearly all cells contain discrete elemental Ca2� signal-ing units, which build up an exquisite hierarchical and dis-continuous architecture in space, time, and intensity, overscales spanning many orders of magnitude (Fig. 17).

In ventricular myocytes as a model system, the char-acteristic spatial scales of Ca2� signaling include the dis-tance between abutting LCCs and RyRs (12–15 nm) or neigh-boring RyRs (30 nm), the size of a single CRU (100–500 nm),the distance between CRUs organized on the same Z-disk(0.5–1.5 �m), and the interval between adjacent Z-disks (1.8�m) (Fig. 10). A spark spans �2.0 �m, a local abortive Ca2�

wavelet travels 5–10 �m, and a full-fledged Ca2� wavesweeps over the entire cell width (20 �m) and length (100–150 �m). Long-range intercellular CICR communication can

also occur via gap junctions that serve as the Ca2� pathsbetween cells. In the subcellular space delimited by the SRmembrane, a cistern that bears a CRU is typically 30 nmthick and 300–600 nm in diameter (48).

The cellular organization of the Ca2� signaling sys-tem in skeletal muscle follows the same general designbut with important differences. Skeletal CRUs consistof elongated double rows of RyRs (instead of the roundor oval geometry of cardiac CRUs), with some para-junctional RyRs in amphibian skeletal muscle fibers,and the sarcomere length is considerably greater (3.0�m). In DRG sensory neurons, CRUs of RyR3 on sub-surface cisterns are particularly enriched on a single-layered shell at lateral intervals of �1.7 �m; CRUs arealso sprinkled over the entire cytoplasm at a reduceddensity, and void in the nucleus (284). Another distinctand remarkable cellular architecture of CRUs is foundin Xenopus oocytes, where IP3R CRUs are almost ex-clusively packaged in a subsurface band about 6 �mthick, and the outer boundary of the zone is separatedfrom the surface membrane by �6 �m (54).

The temporal scales of cardiac Ca2� dynamics rangefrom 100 �s (the establishment and dissipation of sub-space Ca2� gradients) to submillisecond (LCC open time)to 5–50 ms (RyR open time, sparks, blips, blinks) to 0.1–1 s(Ca2� puffs, global [Ca2�]i transients or waves) and to10 s (Ca2� bursts and glows). Modulation of the Ca2�

signaling by various physiological and pathophysiologicalstimuli occurs over a time scale of seconds to minutes(e.g., fight-or-flight response, exercise). Remodeling of theCa2� signaling system, as those in development, cardiachypertrophy, and heart failure, takes effect over muchlonger time scales, i.e., days and months.

FIG. 17. Ca2� signaling multiscaledin space, time, and amplitude.



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In the dimension of concentration, we now havephysiologically relevant cytosolic [Ca2�]i levels in threedifferent zones, spanning at least three orders of mag-nitude. [Ca2�]subspace during Ca2� sparklets and sparksis expected to be as high as a few hundred micromolar,be confined to a volume of 0.002 fl, and to last 1–10 ms.[Ca2�]i in the close atmosphere of a spark is on theorder of 1–10 �M and to spread over a sphere with aradius of 0.1– 0.5 �m in the immediate vicinity of thesubspace. Bulk cytosolic [Ca2�]i is 0.1 �M at rest andvaries by �10-fold during a [Ca2�]i transient. The mi-crodomain [Ca2�] in a spark thus extends the dynamicrange of Ca2� signaling by about an order of magnitude;formation of junctional subspace extends it by yet an-other order of magnitude.

D. The Digital-Analog Dichotomy

Cumulative advances over the last 15 years or sohave led to the suggestion that there is a “digital-analog”duality of Ca2� signaling. In this scenario, discrete Ca2�

microdomains (e.g., those of CRUs) form a digital Ca2�

signaling subsystem that is intertwined with an analogglobal Ca2� signaling subsystem. The digital subsystem ismainly built for high-threshold Ca2�-dependent processes(such as activation of BKCa and ClCa, arrayed RyR, acti-vation of CaMKII and calcineurin, and vesicle secretion).These processes are discontinuous in space and operateintermittently in coincidence with gating of the Ca2� sig-naling units. The autonomy of elemental signaling eventsfurther indicates that they operate essentially in the fre-quency-dependent modulation (FM) mode, though sto-chastic variations are intrinsic to the location, timing, andduration of CRU activation. Hence, low-threshold Ca2�

signaling processes are turned on by bulk Ca2�. Ca2�

signaling should be considered as an analog function,S([Ca2�], t), and high-threshold Ca2� signaling is thusmore appropriately depicted by a digital function, S(nCRU,tevent), where nCRU and tevent denote the number and timeof CRU activation.

E. Biochemistry of Ca2� Sparks

At the molecular level, the basic logic of Ca2� signal-ing is the association and dissociation of a Ca2� to andfrom an effector protein. The Ca2� binding either acti-vates or inhibits a physiological function of the effector,and the Ca2� unbinding either deactivates or disinhibitsthe function. If an effector protein harbors two or morefunctionally opposing Ca2�-binding motifs, interplay be-tween these motifs can render bell-shaped [Ca2�] depen-dence and biphasic kinetics, such that maximal biologicalactivity is attained at an intermediate [Ca2�] over anoptimal time window. At the level of the Ca2� signaling

proteome, the Kd values of Ca2�-binding proteins varyover 7 logarithmic units, ranging from nanomolar to 10mM with a broad mode around 10 �M (Fig. 18), indicatingthe ability of signal transduction at high (�10 �M), inter-mediate (�1 �M), and low Ca2� concentrations (�0.1�M). At the cellular level, the rise and fall of [Ca2�] indifferent compartments are decoded by the vast panel ofCa2� signaling proteins.

Focusing on an elementary event of Ca2� signaling,what biochemical consequences might we expect of asingle Ca2� spark? Or, to what degree is an effectorprotein activated or inactivated inside the Ca2� microdo-main of a spark? Inside the subspace with 100 �M [Ca2�],Ca2� binding sites with a Kon as slow as 5 �M�1 �s�1

(resembling those of EGTA) are occupied by 63% withinas brief a time as 2 ms; moderately fast Ca2� sites areessentially saturated in a spark. It follows that virtually allphysiological relevant Ca2� sites inside the subspace areoccupied during a single Ca2� spark. In the region of aspark, where local [Ca2�] is �1 �M lasting for10 ms, Ca2�

sites with a diffusion-limited Kon (�100 �M�1 �s�1 orfaster) should be substantially occupied; those with 10times slower kinetics (Kon � 10 �M�1 �s�1) should bemildly activated (�10%), whereas those with an evensmaller Kon should be spared. Thus high-, intermediate-,and low-threshold Ca2� signaling processes are likelyactivated in appropriate [Ca2�] zones of a spark. It mightbe of interest to test whether Ca2� effector proteins aredifferentially distributed in the three [Ca2�] zones accord-ing to their association and dissociation kinetics (72). Viaintracellular trafficking, the spark-activated signaling mol-ecules may then broadcast to downstream effectors somedistance away, even those inside the nucleus. Further-more, concurrent signaling processes may occur in thecorresponding cisternal lumen because of deactivation of

FIG. 18. Diversity of Ca2� binding affinity of Ca2�-sensing proteins.The dissociation constants (Kd) of 68 Ca2� binding proteins were ob-tained by text search and manual collection from the literature. [Modi-fied from Cheng et al. (72).]

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effector proteins in a Ca2� blink. If spark-mark couplingcan be further established with direct evidence, mito-chondrial uptake and retention of the spark Ca2� wouldinitiate a large array of signaling cascades that are vital tobioenergetics, ROS metabolism, and the regulation of cellfate. Table 10 summarizes a range of modalities of nano-scopic and microscopic Ca2� signaling with examplesdiscussed in this review.

F. Building Complexity From Simplicity

From a single-atom messenger connecting thousandsof signaling proteins, the Ca2� signaling system is argu-ably the most fascinating biological signal transductionnetwork known. Characterization of the family of elemen-tary Ca2� signaling events has unveiled a “digital-analog”dichotomy and an amazing richness in Ca2� signalingmodalities. Key features of the digital subsystem includediscreteness of CRU organization, local control of CRUsignaling, autonomous CRU operation (with stochasticvariability), nonlinear responsiveness, and FM coding anddecoding of biological information. Building on these,hierarchical structures of Ca2� dynamics emerge, ranging

from sparklets, sparks, and compound sparks, to abortivewavelets and full-fledged propagating Ca2� waves, to spi-ral Ca2� waves that encompass multiscaled spatiotempo-ral substructures (106, 107, 177, 218, 231). Furthermore,local control of Ca2� signaling applies to subcellular com-partments. Concomitant with sparks in the cytosol, thepresence of blinks in the jSR cisternae and marks in singlemitochondria suggests an ability of sparks to coordinatedistinct yet closely related biological functions in differ-ent cellular organelles. These experimental and concep-tual advances put us in a good position to unlock thesecrets underlying Ca2� signaling complexity.

XIX. PERSPECTIVE

Visualization of microdomain Ca2� emanating from asingle or a group of Ca2�-permeant channels in intactcells has electrified the investigation of Ca2� signaling indiverse physiological processes such as EC coupling, vas-cular tone regulation, pacemaker activity, modulation ofexcitability, and exocytosis. Characterization of a familyof elementary Ca2� signaling events, exemplified by Ca2�

sparks, has uncovered a digital Ca2� signaling subsystemthat builds on discrete, stochastic, and autonomousevents and displays exquisite architecture in space, time,and intensity over many orders of magnitude. Character-ization of sparks has also provided mechanistic insightsinto the development of a variety of diseases in cardiac,skeletal, and smooth muscles. These advances not onlyresolve old paradoxes, but also promise to unify andsimplify the complexity of Ca2� signaling.

Despite a wealth of information, many importantquestions of sparkology remain to be answered. For in-stance, the nature of Ca2� sparks is still not fully under-stood. At the heart of the question is the enigmatic be-havior of arrayed RyRs in intact cells. Do RyRs in a CRUact in unison, or do they operate in dynamic subgroups?What are the fates of individual RyRs in a CRU during aspark? What mechanisms are responsible for the termi-nation of array activation? We are only beginning to ap-preciate the unique biophysics and physiology of CRUoperation in cells.

CRUs, the hubs of the digital Ca2� signaling sub-system, are naturally subjected to multilayered physiolog-ical regulation. A myriad of Ca2� signaling proteins formmacromolecular complexes with the release channels, onboth the cytosolic and the luminal sides as well as in theSR membrane. Much is to be learned about how therelease channels are modulated by the large panel ofmolecular partners. From a broader cell biological view-point, it is also of fundamental importance to understandthe trafficking, assembly, maintenance, and turnover ofCRUs, couplons and other supramolecular nanomachinesof Ca2� signaling.

TABLE 10. Modalities of local Ca2� signaling

Examples

Intramolecular 1) The rise of �Ca2�� at the mouth of a RyRcauses Ca2�-dependent activation orinactivation of the channel (422).

2) High �Ca2�� at the mouth of a Ca2�

channel induces Ca2�-dependentfacilitation and inactivation of thechannel (64, 104, 265).

Intra-CRU 1) Putative CICR or Ca2�-dependentinactivation among RyRs in the sameCRUs on the cytosolic side.

2) Communication between junctionalRyR1 array and parajunctional RyR3array in an amphibian skeletal musclecouplon (300).

3) Putative desensitization of a CRU due tocisternal Ca2� depletion in a spark.

Inter-CRU Compound sparks; Ca2� wavelets; Ca2�

waves of spheric, planar, or spiralwavefronts; perinuclear Ca2� waves.

Ca2� synapses 1) Single-channel LCC currents activatesparks in a cardiac CRU (390).

2) Spark activates a BKCa STOC or ClCa

STIC (274, 446).3) Spark modulation of exocytosis in DRG

neurons (284).4) Tunneling the ER/SR released Ca2� to

mitochondria (337).Others 1) Ca2� entry of VDCCs activates SKCa on

the plasma membrane (164).2) Store depletion causes STIM1 and STIM2

to aggregate and then translocate tospecialized subsurface ER elementswhere they activate Orai1, a prototypicstore-operated Ca2� channel (43, 227).



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The recent demonstration of nanoscopic Ca2� sig-nals inside the SR and other cellular organelles representsan exciting development, for it illustrates how cytosolicmicrodomain Ca2� is coordinated with Ca2� in topologi-cally segregated subcellular compartments. The technicaladvances with genetically encoded, organelle- or intramo-lecular-targeted Ca2� indicators have paved the way to-ward new horizons of local Ca2� signaling.

Ca2� sparks and other elemental signals provide in-vestigative tools with multiple facets. Diffusion-limited insize and efficacious for high-threshold Ca2�-dependentprocesses, they can be exploited to probe physiologicaland pathophysiological processes at unprecedented pre-cision. Exemplary cases include sparklet-spark coupling,spark-STOC or STIC coupling, spark-NCX coupling incardiac pacemaker and atrial cells, spark-mark coupling,and spark-secretion coupling in neurons. We also begin toappreciate the prominent biological role of Ca2� clocks inmany types of cells. Efforts should be devoted to theelucidation of microdomain Ca2� signals in long-termregulation of cellular functions, including excitation-tran-scription coupling, hypertrophy, and apoptosis.

For the Ca2� signaling system as a whole, we nowhave a glimpse of its organizational principles decipheredfrom the exquisite spatial, temporal, and concentrationarchitecture. However, little is known about how Ca2�

sparks and other elemental signals direct the biologicalinformation across a network of thousands of proteins.Advances in bioinformatics and systems biology are nowforging novel tools to tackle these formidable questions. Itremains a physiologist’s wildest dream to understand thebiological wisdom of building the greatest complexityfrom the utmost simplicity.

ACKNOWLEDGMENTS

We thank Iain Bruce, Pamela Wright, Sheng Wei, Ting Zhao,Huaqiang Fang, and Yuan Yan for editorial assistance. We are indebt to our colleagues, postdoctoral fellows, and students whoover the past 15 years have made great contributions to thework discussed in this review. We are also grateful to the manyinvestigators who made their most recent progress available tothis review.

Address for reprint requests and other correspondence: H.Cheng, Laboratory of Calcium Signaling, Institute of MolecularMedicine, Peking University, Beijing 100871, China (e-mail:[email protected]; [email protected]).

GRANTS

This work was supported by Chinese National NaturalScience Funds (30630021), Major State Basic Research Devel-opment Programs (2007CB512100; to H. Cheng), NationalHeart, Lung, and Blood Institute, Fondation Leducq, and Stateof Maryland Stem Cell Program (to W. J. Lederer; [email protected]).

REFERENCES

1. Abriel H, Cabo C, Wehrens XH, Rivolta I, Motoike HK,

Memmi M, Napolitano C, Priori SG, Kass RS. Novel arrhyth-mogenic mechanism revealed by a long-QT syndrome mutation inthe cardiac Na� channel. Circ Res 88: 740–745, 2001.

2. Allen DG, Blinks JR. Calcium transients in aequorin-injected frogcardiac muscle. Nature 273: 509–513, 1978.

3. Altamirano J, Li Y, Desantiago J, Piacentino V III, Houser SR,

Bers DM. The inotropic effect of cardioactive glycosides in ven-tricular myocytes requires Na�-Ca2� exchanger function. J Physiol

575: 845–854, 2006.4. Amberg GC, Bonev AD, Rossow CF, Nelson MT, Santana LF.

Modulation of the molecular composition of large conductance,Ca2� activated K� channels in vascular smooth muscle duringhypertension. J Clin Invest 112: 717–724, 2003.

5. Amberg GC, Navedo MF, Nieves-Cintron M, Molkentin JD,

Santana LF. Calcium sparklets regulate local and global calciumin murine arterial smooth muscle. J Physiol 579: 187–201, 2007.

6. Amberg GC, Santana LF. Downregulation of the BK channelbeta1 subunit in genetic hypertension. Circ Res 93: 965–971, 2003.

7. Ashley CC, Ridgway EB. On the relationships between membranepotential, calcium transient and tension in single barnacle musclefibres. J Physiol 209: 105–130, 1970.

8. Balasubramanian L, Ahmed A, Lo CM, Sham JSK, Yip KP.

Integrin-mediated mechanotransduction in renal vascular smoothmuscle cells: activation of calcium sparks. Am J Physiol Regul

Integr Comp Physiol 293: R1586–R1594, 2007.9. Balijepalli RC, Lokuta AJ, Maertz NA, Buck JM, Haworth RA,

Valdivia HH, Kamp TJ. Depletion of T-tubules and specific sub-cellular changes in sarcolemmal proteins in tachycardia-inducedheart failure. Cardiovasc Res 59: 67–77, 2003.

10. Ballester LY, Benson DW, Wong B, Law IH, Mathews KD,

Vanoye CG, George AL Jr. Trafficking-competent and trafficking-defective KCNJ2 mutations in Andersen syndrome. Hum Mutat 27:388, 2006.

11. Banyasz T, Chen-Izu Y, Balke CW, Izu LT. A new approach tothe detection and statistical classification of Ca2� sparks. Biophys

J 92: 4458–4465, 2007.12. Bao L, Jin SX, Zhang C, Wang LH, Xu ZZ, Zhang FX, Wang LC,

Ning FS, Cai HJ, Guan JS, Xiao HS, Xu ZQ, He C, Hokfelt T,

Zhou Z, Zhang X. Activation of delta opioid receptors inducesreceptor insertion and neuropeptide secretion. Neuron 37: 121–133,2003.

13. Barac YD, Zeevi-Levin N, Yaniv G, Reiter I, Milman F, Shilkrut

M, Coleman R, Abassi Z, Binah O. The 1,4,5-inositol trisphos-phate pathway is a key component in Fas-mediated hypertrophy inneonatal rat ventricular myocytes. Cardiovasc Res 68: 75–86, 2005.

14. Bare DJ, Kettlun CS, Liang M, Bers DM, Mignery GA. Cardiactype 2 inositol 1,4,5-trisphosphate receptor: interaction and modu-lation by calcium/calmodulin-dependent protein kinase II. J Biol

Chem 280: 15912–15920, 2005.15. Baylor SM. Calcium sparks in skeletal muscle fibers. Cell Calcium

37: 513–530, 2005.16. Baylor SM, Hollingworth S, Chandler WK. Comparison of sim-

ulated and measured calcium sparks in intact skeletal muscle fibersof the frog. J Gen Physiol 120: 349–368, 2002.

17. Beard NA, Casarotto MG, Wei L, Varsanyi M, Laver DR,

Dulhunty AF. Regulation of ryanodine receptors by calsequestrin:effect of high luminal Ca2� and phosphorylation. Biophys J 88:3444–3454, 2005.

18. Bellinger AM, Reiken S, Dura M, Murphy PW, Deng SX,

Landry DW, Nieman D, Lehnart SE, Samaru M, Lacampagne

A, Marks AR. Remodeling of ryanodine receptor complex causes“leaky” channels: a molecular mechanism for decreased exercisecapacity. Proc Natl Acad Sci USA 105: 2198–2202, 2008.

19. Benham CD, Bolton TB. Spontaneous transient outward currentsin single visceral and vascular smooth-muscle cells of the rabbit.J Physiol 381: 385–406, 1986.

20. Berlin JR, Cannell MB, Lederer WJ. Cellular origins of thetransient inward current in cardiac myocytes. Role of fluctuationsand waves of elevated intracellular calcium. Circ Res 65: 115–126,1989.

CALCIUM SPARKS 1535


on May 25, 2011


ownloaded from


21. Berridge MJ. Inositol trisphosphate and calcium signalling.Nature 361: 315–325, 1993.

22. Berridge MJ. Neuronal calcium signaling. Neuron 21: 13–26, 1998.23. Berridge MJ. The endoplasmic reticulum: a multifunctional sig-

naling organelle. Cell Calcium 32: 235–249, 2002.24. Berridge MJ. Calcium microdomains: organization and function.

Cell Calcium 40: 405–412, 2006.25. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:

dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529, 2003.

26. Berridge MJ, Lipp P, Bootman MD. The versatility and univer-sality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000.

27. Bers DM. Dynamic imaging in living cells: windows into localsignaling. Sci STKE 2003: e13, 2003.

28. Bers DM. Excitation-Contraction Coupling and Cardiac Con-

tractile Force (2nd ed.). Netherlands: Kluwer Academic, 2001.29. Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca2�

and heart failure: roles of diastolic leak and Ca2� transport. Circ

Res 93: 487–490, 2003.30. Bers DM, Fill M. Coordinated feet and the dance of ryanodine

receptors. Science 281: 790–791, 1998.31. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych

S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess

HF. Imaging intracellular fluorescent proteins at nanometer reso-lution. Science 313: 1642–1645, 2006.

32. Bhat MB, Hayek SM, Zhao J, Zang W, Takeshima H, Wier WG,

Ma J. Expression and functional characterization of the cardiacmuscle ryanodine receptor Ca2� release channel in Chinese ham-ster ovary cells. Biophys J 77: 808–816, 1999.

33. Bhat MB, Zhao J, Zang W, Balke CW, Takeshima H, Wier WG,

Ma J. Caffeine-induced release of intracellular Ca2� from Chinesehamster ovary cells expressing skeletal muscle ryanodine receptor.Effects on full-length and carboxyl-terminal portion of Ca2� releasechannels. J Gen Physiol 110: 749–762, 1997.

34. Blinks JR, Prendergast FG, Allen DA. Photoproteins as biolog-ical calcium indicators. Pharmacol Rev 28: 1–93, 1976.

35. Bogdanov KY, Maltsev VA, Vinogradova TM, Lyashkov AE,

Spurgeon HA, Stern MD, Lakatta EG. Membrane potential fluc-tuations resulting from submembrane Ca2� releases in rabbit sino-atrial nodal cells impart an exponential phase to the late diastolicdepolarization that controls their chronotropic state. Circ Res 99:979–987, 2006.

36. Bogdanov KY, Vinogradova TM, Lakatta EG. Sinoatrial nodalcell ryanodine receptor and Na�-Ca2� exchanger: molecular part-ners in pacemaker regulation. Circ Res 88: 1254–1258, 2001.

37. Bolton TB, Imaizumi Y. Spontaneous transient outward currentsin smooth muscle cells. Cell Calcium 20: 141–152, 1996.

38. Bolton TB, Prestwich SA, Zholos AV, Gordienko DV. Excita-tion-contraction coupling in gastrointestinal and other smoothmuscles. Annu Rev Physiol 61: 85–115, 1999.

39. Bond RA, Leff P, Johnson TD, Milano CA, Rockman HA,

Mcminn TR, Apparsundaram S, Hyek MF, Kenakin TP, Allen

LF, Lefkowitz RJ. Physiological effects of inverse agonists intransgenic mice with myocardial overexpression of the beta 2-ad-renoceptor. Nature 374: 272–276, 1995.

40. Bootman M, Niggli E, Berridge M, Lipp P. Imaging the hierar-chical Ca2� signalling system in HeLa cells. J Physiol 499: 307–314,1997.

41. Bootman MD, Berridge MJ. The elemental principles of calciumsignaling. Cell 83: 675–678, 1995.

42. Borisova L, Shmygol A, Wray S, Burdyga T. Evidence that aCa2� sparks/STOCs coupling mechanism is responsible for theinhibitory effect of caffeine on electro-mechanical coupling inguinea pig ureteric smooth muscle. Cell Calcium 42: 303–311, 2007.

43. Brandman O, Liou J, Park WS, Meyer T. STIM2 is a feedbackregulator that stabilizes basal cytosolic and endoplasmic reticulumCa2� levels. Cell 131: 1327–1339, 2007.

44. Bray MA, Geisse NA, Parker KK. Multidimensional detectionand analysis of Ca2� sparks in cardiac myocytes. Biophys J 92:4433–4443, 2007.

45. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC,

Wiler SW, Patterson AJ, Nelson MT, Aldrich RW. Vasoregula-

tion by the beta1 subunit of the calcium-activated potassium chan-nel. Nature 407: 870–876, 2000.

46. Bridge JH, Ershler PR, Cannell MB. Properties of Ca2� sparksevoked by action potentials in mouse ventricular myocytes.J Physiol 518: 469–478, 1999.

47. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova

E, Moschella MC, Jayaraman T, Landers M, Ehrlich BE,

Marks AR. Stabilization of calcium release channel (ryanodinereceptor) function by FK506-binding protein. Cell 77: 513–523,1994.

48. Brochet DX, Yang D, Di MA, Lederer WJ, Franzini-Armstrong

C, Cheng H. Ca2� blinks: rapid nanoscopic store calcium signal-ing. Proc Natl Acad Sci USA 102: 3099–3104, 2005.

49. Brown JE, Cohen LB, De WP, Pinto LH, Ross WN, Salzberg

BM. Rapid changes in intracellular free calcium concentration.Detection by metallochromic indicator dyes in squid giant axon.Biophys J 15: 1155–1160, 1975.

50. Brown LD, Rodney GG, Hernandez-Ochoa E, Ward CW,

Schneider MF. Ca2� sparks and T tubule reorganization in dedi-fferentiating adult mouse skeletal muscle fibers. Am J Physiol Cell

Physiol 292: C1156–C1166, 2007.51. Brum G, Gonzalez A, Rengifo J, Shirokova N, Rios E. Fast

imaging in two dimensions resolves extensive sources of Ca2�

sparks in frog skeletal muscle. J Physiol 528: 419–433, 2000.52. Burdyga T, Wray S. Action potential refractory period in ureter

smooth muscle is set by Ca sparks and BK channels. Nature 436:559–562, 2005.

53. Callamaras N, Parker I. Ca2�-dependent activation of Cl� cur-rents in Xenopus oocytes is modulated by voltage. Am J Physiol

Cell Physiol 278: C667–C675, 2000.54. Callamaras N, Parker I. Radial localization of inositol 1,4,5-

trisphosphate-sensitive Ca2� release sites in Xenopus oocytes re-solved by axial confocal linescan imaging. J Gen Physiol 113:199–213, 1999.

55. Callamaras N, Parker I. Phasic characteristic of elementary Ca2�

release sites underlies quantal responses to IP3. EMBO J 19: 3608–3617, 2000.

56. Cannell MB, Berlin JR, Lederer WJ. Intracellular calcium incardiac myocytes: calcium transients measured using fluorescenceimaging. Soc Gen Physiol Ser 42: 201–214, 1987.

57. Cannell MB, Cheng H, Lederer WJ. The control of calciumrelease in heart muscle. Science 268: 1045–1049, 1995.

58. Cannell MB, Cheng H, Lederer WJ. Spatial non-uniformities in[Ca2�]i during excitation-contraction coupling in cardiac myocytes.Biophys J 67: 1942–1956, 1994.

59. Cannell MB, Crossman DJ, Soeller C. Effect of changes inaction potential spike configuration, junctional sarcoplasmic retic-ulum micro-architecture and altered t-tubule structure in humanheart failure. J Muscle Res Cell Motil 27: 297–306, 2006.

60. Cannell MB, Lederer WJ. The arrhythmogenic current ITI in theabsence of electrogenic sodium-calcium exchange in sheep cardiacPurkinje fibres. J Physiol 374: 201–219, 1986.

61. Cannell MB, Soeller C. Numerical analysis of ryanodine receptoractivation by L-type channel activity in the cardiac muscle diad.Biophys J 73: 112–122, 1997.

62. Capogrossi MC, Stern MD, Spurgeon HA, Lakatta EG. Spon-taneous Ca2� release from the sarcoplasmic reticulum limits Ca2�-dependent twitch potentiation in individual cardiac myocytes.J Gen Physiol 91: 133–155, 1988.

63. Chandler WK, Hollingworth S, Baylor SM. Simulation of cal-cium sparks in cut skeletal muscle fibers of the frog. J Gen Physiol

121: 311–324, 2003.64. Chaudhuri D, Issa JB, Yue DT. Elementary mechanisms produc-

ing facilitation of Ca(v)2.1 (P/Q-type) channels. J Gen Physiol 129:385–401, 2007.

65. Cheng H, Cannell MB, Lederer WJ. Partial inhibition of Ca2�

current by methoxyverapamil (D600) reveals spatial nonuniformi-ties in [Ca2�]i during excitation-contraction coupling in cardiacmyocytes. Circ Res 76: 236–241, 1995.

66. Cheng H, Fill M, Valdivia H, Lederer WJ. Models of Ca2� releasechannel adaptation. Science 267: 2009–2010, 1995.



on May 25, 2011


ownloaded from


67. Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calciumsparks and [Ca2�]i waves in cardiac myocytes. Am J Physiol Cell

Physiol 270: C148–C159, 1996.68. Cheng H, Lederer MR, Xiao RP, Gomez AM, Zhou YY, Ziman B,

Spurgeon H, Lakatta EG, Lederer WJ. Excitation-contractioncoupling in heart: new insights from Ca2� sparks. Cell Calcium 20:129–140, 1996.

69. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementaryevents underlying excitation-contraction coupling in heart muscle.Science 262: 740–744, 1993.

70. Cheng H, Song LS, Shirokova N, Gonzalez A, Lakatta EG,

Rios E, Stern MD. Amplitude distribution of calcium sparks inconfocal images: theory and studies with an automatic detectionmethod. Biophys J 76: 606–617, 1999.

71. Cheng H, Wang SQ. Calcium signaling between sarcolemmal cal-cium channels and ryanodine receptors in heart cells. Front Biosci

7: d1867–d1878, 2002.72. Cheng HP, Wei S, Wei LP, Verkhratsky A. Calcium signaling in

physiology and pathophysiology. Acta Pharmacol Sin 27: 767–772,2006.

73. Choi BR, Burton F, Salama G. Cytosolic Ca2� triggers earlyafterdepolarizations and Torsade de Pointes in rabbit hearts withtype 2 long QT syndrome. J Physiol 543: 615–631, 2002.

75. Choi KM, Zhong Y, Hoit BD, Grupp IL, Hahn H, Dilly KW,

Guatimosim S, Lederer WJ, Matlib MA. Defective intracellularCa2� signaling contributes to cardiomyopathy in Type 1 diabeticrats. Am J Physiol Heart Circ Physiol 283: H1398–H1408, 2002.

76. Chopra N, Kannankeril PJ, Yang T, Hlaing T, Holinstat I,

Ettensohn K, Pfeifer K, Akin B, Jones LR, Franzini-Arm-

strong C, Knollmann BC. Modest reductions of cardiac calse-questrin increase sarcoplasmic reticulum Ca2� leak independent ofluminal Ca2� and trigger ventricular arrhythmias in mice. Circ Res

101: 617–626, 2007.77. Chow RH, Klingauf J, Neher E. Time course of Ca2� concentra-

tion triggering exocytosis in neuroendocrine cells. Proc Natl Acad

Sci USA 91: 12765–12769, 1994.78. Clancy CE, Kass RS. Inherited and acquired vulnerability to ven-

tricular arrhythmias: cardiac Na� and K� channels. Physiol Rev 85:33–47, 2005.

79. Clapham DE. Calcium signaling. Cell 131: 1047–1058, 2007.80. Cleemann L, DiMassa G, Morad M. Ca2� sparks within 200 nm of

the sarcolemma of rat ventricular cells: evidence from total internalreflection fluorescence microscopy. Adv Exp Med Biol 430: 57–65,1997.

81. Cleemann L, Wang W, Morad M. Two-dimensional confocal im-ages of organization, density, gating of focal Ca2� release sites inrat cardiac myocytes. Proc Natl Acad Sci USA 95: 10984–10989,1998.

82. Clusin WT. Calcium and cardiac arrhythmias: DADs, EADs, alter-nans. Crit Rev Clin Lab Sci 40: 337–375, 2003.

83. Clusin WT, Buchbinder M, Harrison DC. Calcium overload,“injury” current, early ischaemic cardiac arrhythmias—a directconnection. Lancet 1: 272–274, 1983.

84. Collier ML, Ji G, Wang Y, Kotlikoff MI. Calcium-induced cal-cium release in smooth muscle: loose coupling between the actionpotential and calcium release. J Gen Physiol 115: 653–662, 2000.

85. Conklin MW, Ahern CA, Vallejo P, Sorrentino V, Takeshima

H, Coronado R. Comparison of Ca2� sparks produced indepen-dently by two ryanodine receptor isoforms (type 1 or type 3).Biophys J 78: 1777–1785, 2000.

86. Conklin MW, Barone V, Sorrentino V, Coronado R. Contribu-tion of ryanodine receptor type 3 to Ca2� sparks in embryonicmouse skeletal muscle. Biophys J 77: 1394–1403, 1999.

87. Coombes S, Hinch R, Timofeeva Y. Receptors, sparks and wavesin a fire-diffuse-fire framework for calcium release. Prog Biophys

Mol Biol 85: 197–216, 2004.88. Copello JA, Zima AV, az-Sylvester PL, Fill M, Blatter LA. Ca2�

entry-independent effects of L-type Ca2� channel modulators onCa2� sparks in ventricular myocytes. Am J Physiol Cell Physiol

292: C2129–C2140, 2007.89. Cordeiro JM, Bridge JH, Spitzer KW. Early and delayed after-

depolarizations in rabbit heart Purkinje cells viewed by confocalmicroscopy. Cell Calcium 29: 289–297, 2001.

90. Csernoch L. Sparks and embers of skeletal muscle: the excitingevents of contractile activation. Pflugers Arch 454: 869–878, 2007.

91. Csernoch L, Zhou J, Stern MD, Brum G, Rios E. The elementaryevents of Ca2� release elicited by membrane depolarization inmammalian muscle. J Physiol 557: 43–58, 2004.

92. Damiano BP, Rosen MR. Effects of pacing on triggered activityinduced by early afterdepolarizations. Circulation 69: 1013–1025,1984.

93. Dash R, Frank KF, Carr AN, Moravec CS, Kranias EG. Genderinfluences on sarcoplasmic reticulum Ca2�-handling in failing hu-man myocardium. J Mol Cell Cardiol 33: 1345–1353, 2001.

94. Davidoff AJ. Convergence of glucose- and fatty acid-induced ab-normal myocardial excitation-contraction coupling and insulin sig-nalling. Clin Exp Pharmacol Physiol 33: 152–158, 2006.

95. De Crescenzo V, ZhuGe R, Velazquez-Marrero C, Lifshitz LM,

Custer E, Carmichael J, Lai FA, Tuft RA, Fogarty KE, Lemos

JR, Walsh JV Jr. Ca2� syntillas, miniature Ca2� release events interminals of hypothalamic neurons, are increased in frequency bydepolarization in the absence of Ca2� influx. J Neurosci 24: 1226–1235, 2004.

96. DelPrincipe F, Egger M, Ellis-Davies GC, Niggli E. Two-photonand UV-laser flash photolysis of the Ca2� cage, dimethoxynitrophe-nyl-EGTA-4. Cell Calcium 25: 85–91, 1999.

97. DelPrincipe F, Egger M, Niggli E. Calcium signalling in cardiacmuscle: refractoriness revealed by coherent activation. Nat Cell

Biol 1: 323–329, 1999.98. Demuro A, Parker I. “Optical patch-clamping”: single-channel

recording by imaging Ca2� flux through individual muscle acetyl-choline receptor channels. J Gen Physiol 126: 179–192, 2005.

99. Demuro A, Parker I. Imaging single-channel calcium microdo-mains. Cell Calcium 40: 413–422, 2006.

100. Demuro A, Parker I. Optical single-channel recording: imagingCa2� flux through individual N-type voltage-gated channels ex-pressed in Xenopus oocytes. Cell Calcium 34: 499–509, 2003.

101. Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM. Intra-cellular Na� concentration is elevated in heart failure but Na/Kpump function is unchanged. Circulation 105: 2543–2548, 2002.

102. Devine CE, Somlyo AV, Somlyo AP. Sarcoplasmic reticulum andexcitation-contraction coupling in mammalian smooth muscles.J Cell Biol 52: 690, 1972.

103. Diaz ME, O’Neill SC, Eisner DA. Sarcoplasmic reticulum cal-cium content fluctuation is the key to cardiac alternans. Circ Res

94: 650–656, 2004.104. Dick IE, Tadross MR, Liang HY, Tay LH, Yang WJ, Yue DT. A

modular switch for spatial Ca2� selectivity in the calmodulin reg-ulation of Ca-V channels. Nature 451: 830–879, 2008.

105. Dirksen WP, Lacombe VA, Chi M, Kalyanasundaram A, Viatch-

enko-Karpinski S, Terentyev D, Zhou ZX, Vedamoorthyrao S,

Li N, Chiamvimonvat N, Carnes CA, Franzini-Armstrong C,

Gyorke S, Periasamy M. A mutation in calsequestrin, CASQ2(D307H), impairs sarcoplasmic reticulum Ca2� handling andcauses complex ventricular arrhythmias in mice. Cardiovasc Res

75: 69–78, 2007.106. Dupont G. Theoretical insights into the mechanism of spiral Ca2�

wave initiation in Xenopus oocytes. Am J Physiol Cell Physiol 275:C317–C322, 1998.

107. Dupont G, Pontes J, Goldbeter A. Modeling spiral Ca2� waves insingle cardiac cells: role of the spatial heterogeneity created by thenucleus. Am J Physiol Cell Physiol 271: C1390–C1399, 1996.

108. Eisner DA, Diaz ME, Li Y, O’Neill SC, Trafford AW. Stabilityand instability of regulation of intracellular calcium. Exp Physiol

90: 3–12, 2005.109. Eisner DA, Lederer WJ. The role of the sodium pump in the

effects of potassium-depleted solutions on mammalian cardiacmuscle. J Physiol 294: 279–301, 1979.

110. Eisner DA, Lederer WJ, Vaughan-Jones RD. The dependence ofsodium pumping and tension on intracellular sodium activity involtage-clamped sheep Purkinje fibres. J Physiol 317: 163–187,1981.

111. Eisner DA, Trafford AW, Diaz ME, Overend CL, O’Neill SC.

The control of Ca release from the cardiac sarcoplasmic reticulum:regulation versus autoregulation. Cardiovasc Res 38: 589–604,1998.

CALCIUM SPARKS 1537


on May 25, 2011


ownloaded from


112. Endo M, Tanaka M, Ogawa Y. Calcium induced release of cal-cium from sarcoplasmic reticulum of skinned skeletal musclefibres. Nature 228: 34, 1970.

113. Essin K, Welling A, Hofmann F, Luft FC, Gollasch M, Moos-

mang S. Indirect coupling between Ca(v)1.2 channels and ryano-dine receptors to generate Ca2� sparks in murine arterial smoothmuscle cells. J Physiol 584: 205–219, 2007.

114. Fabiato A. Rapid ionic modifications during the aequorin-detectedcalcium transient in a skinned canine cardiac Purkinje cell. J Gen

Physiol 85: 189–246, 1985.115. Fabiato A. Simulated calcium current can both cause calcium

loading in and trigger calcium release from the sarcoplasmic retic-ulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85:291–320, 1985.

116. Fabiato A. Time and calcium dependence of activation and inac-tivation of calcium-induced release of calcium from the sarcoplas-mic reticulum of a skinned canine cardiac Purkinje cell. J Gen

Physiol 85: 247–289, 1985.117. Felder E, Franzini-Armstrong C. Type 3 ryanodine receptors of

skeletal muscle are segregated in a parajunctional position. Proc

Natl Acad Sci USA 99: 1695–1700, 2002.118. Ferrier GR, Moe GK. Effect of calcium on acetylstrophanthidin-

induced transient depolarizations in canine purkinje tissue. Circ

Res 33: 508–515, 1973.119. Ferrier GR, Saunders JH, Mendez C. A cellular mechanism for

the generation of ventricular arrhythmias by acetylstrophanthidin.Circ Res 32: 600–609, 1973.

120. Ferris CD, Cameron AM, Huganir RL, Snyder SH. Quantalcalcium release by purified reconstituted inositol 1,4,5-triphos-phate receptors. Nature 356: 350–352, 1992.

121. Fill M, Copello JA. Ryanodine receptor calcium release channels.Physiol Rev 82: 893–922, 2002.

122. Fill M, Zahradnikova A, Villalba-Galea CA, Zahradnik I,

Escobar AL, Gyorke S. Ryanodine receptor adaptation. J Gen

Physiol 116: 873–882, 2000.123. Flaim SN, Giles WR, McCulloch AD. Arrhythmogenic conse-

quences of Na� channel mutations in the transmurally heteroge-neous mammalian left ventricle: analysis of the I1768V SCN5Amutation. Heart Rhythm 4: 768–778, 2007.

124. Ford LE, Podolsky RJ. Regenerative calcium release within mus-cle cells. Science 167: 58, 1970.

125. Foskett JK, White C, Cheung KH, Mak DOD. Inositol trisphos-phate receptor Ca2� release channels. Physiol Rev 87: 593–658,2007.

126. Franzini-Armstrong C. Structure of sarcoplasmic reticulum. Fed-

eration Proc 39: 2403–2409, 1980.127. Franzini-Armstrong C, Kenney LJ, Varriano Marston E. The

structure of calsequestrin in triads of vertebrate skeletal muscle: adeep-etch study. J Cell Biol 105: 49–56, 1987.

128. Franzini-Armstrong C, Protasi F. Ryanodine receptors of stri-ated muscles: a complex channel capable of multiple interactions.Physiol Rev 77: 699–729, 1997.

129. Franzini-Armstrong C, Protasi F, Ramesh V. Comparativeultrastructure of Ca2� release units in skeletal and cardiac muscle.Ann NY Acad Sci 853: 20–30, 1998.

130. Franzini-Armstrong C, Protasi F, Ramesh V. Shape, size, dis-tribution of Ca2� release units and couplons in skeletal and cardiacmuscles. Biophys J 77: 1528–1539, 1999.

131. Franzini-Armstrong C, Protasi F, Tijskens P. The assembly ofcalcium release units in cardiac muscle. Ann NY Acad Sci 1047:76–85, 2005.

132. Fredj S, Lindegger N, Sampson KJ, Carmeliet P, Kass RS.

Altered Na� channels promote pause-induced spontaneous dia-stolic activity in long QT syndrome type 3 myocytes. Circ Res 99:1225–1232, 2006.

133. Furstenau M, Lohn M, Ried C, Luft FC, Haller H, Gollasch M.

Calcium sparks in human coronary artery smooth muscle cellsresolved by confocal imaging. J Hypertens 18: 1215–1222, 2000.

134. Gassanov N, Brandt MC, Michels G, Lindner M, Er F, Hoppe

UC. Angiotensin II-induced changes of calcium sparks and ioniccurrents in human atrial myocytes: potential role for early remod-eling in atrial fibrillation. Cell Calcium 39: 175–186, 2006.

135. Gergs U, Berndt T, Buskase J, Jones LR, Kirchhefer U, Muller

FU, Schluter KD, Schmitz W, Neumann J. On the role of junctinin cardiac Ca2� handling, contractility, heart failure. Am J Physiol

Heart Circ Physiol 293: H728–H734, 2007.136. Gilkey JC, Jaffe LF, Ridgway EB, Reynolds GT. A free calcium

wave traverses the activating egg of the medaka, Oryzias latipes.J Cell Biol 76: 448–466, 1978.

137. Go LO, Moschella MC, Watras J, Handa KK, Fyfe BS, Marks

AR. Differential regulation of two types of intracellular calciumrelease channels during end-stage heart failure. J Clin Invest 95:888–894, 1995.

138. Goldhaber JI, Xie LH, Duong T, Motter C, Khuu K, Weiss JN.

Action potential duration restitution and alternans in rabbit ven-tricular myocytes: the key role of intracellular calcium cycling.Circ Res 96: 459–466, 2005.

139. Gollasch M, Wellman GC, Knot HJ, Jaggar JH, Damon DH,

Bonev AD, Nelson MT. Ontogeny of local sarcoplasmic reticulumCa2� signals in cerebral arteries: Ca2� sparks as elementary phys-iological events. Circ Res 83: 1104–1114, 1998.

140. Gomez TM, Zheng JQ. The molecular basis for calcium-depen-dent axon pathfinding. Nat Rev Neurosci 7: 115–125, 2006.

141. Gonzalez A, Kirsch WG, Shirokova N, Pizarro G, Brum G,

Pessah IN, Stern MD, Cheng H, Rios E. Involvement of multipleintracellular release channels in calcium sparks of skeletal muscle.Proc Natl Acad Sci USA 97: 4380–4385, 2000.

142. Gonzalez A, Kirsch WG, Shirokova N, Pizarro G, Stern MD,

Rios E. The spark and its ember: separately gated local compo-nents of Ca2� release in skeletal muscle. J Gen Physiol 115: 139–158, 2000.

143. Goonasekera SA, Beard NA, Groom L, Kimura T, Lyfenko AD,

Rosenfeld A, Marty I, Dulhunty AF, Dirksen RT. Triadin bind-ing to the C-terminal luminal loop of the ryanodine receptor isimportant for skeletal muscle excitation contraction coupling.J Gen Physiol 130: 365–378, 2007.

144. Gordienko DV, Bolton TB. Crosstalk between ryanodine recep-tors and IP3 receptors as a factor shaping spontaneous Ca2�-release events in rabbit portal vein myocytes. J Physiol 542: 743–762, 2002.

145. Gordienko DV, Bolton TB, Cannell MB. Variability in spontane-ous subcellular calcium release in guinea-pig ileum smooth musclecells. J Physiol 507: 707–720, 1998.

146. Grantham CJ, Cannell MB. Ca2� influx during the cardiac actionpotential in guinea pig ventricular myocytes. Circ Res 79: 194–200,1996.

147. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2�

indicators with greatly improved fluorescence properties. J Biol

Chem 260: 3440–3448, 1985.148. Guatimosim S, Dilly K, Santana LF, Saleet Jafri M, Sobie EA,

Lederer WJ. Local Ca2� signaling and EC coupling in heart: Ca2�

sparks and the regulation of the [Ca2�]i transient. J Mol Cell Car-

diol 34: 941–950, 2002.149. Guia A, Stern MD, Lakatta EG, Josephson IR. Ion concentra-

tion-dependence of rat cardiac unitary L-type calcium channelconductance. Biophys J 80: 2742–2750, 2001.

150. Gyorke I, Gyorke S. Regulation of the cardiac ryanodine receptorchannel by luminal Ca2� involves luminal Ca2� sensing sites. Bio-

phys J 75: 2801–2810, 1998.151. Gyorke I, Hester N, Jones LR, Gyorke S. The role of calseques-

trin, triadin, junctin in conferring cardiac ryanodine receptor re-sponsiveness to luminal calcium. Biophys J 86: 2121–2128, 2004.

152. Gyorke S, Fill M. Ryanodine receptor adaptation: control mech-anism of Ca2�-induced Ca2� release in heart. Science 260: 807–809,1993.

153. Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-

Karpinski S, Wiesner TF. Regulation of sarcoplasmic reticulumcalcium release by luminal calcium in cardiac muscle. Front Biosci

7: d1454–d1463, 2002.154. Gyorke S, Hagen BM, Terentyev D, Lederer WJ. Chain-reaction

Ca2� signaling in the heart. J Clin Invest 117: 1758–1762, 2007.155. Gyorke S, Terentyev D. Modulation of ryanodine receptor by

luminal calcium and accessory proteins in health and cardiac dis-ease. Cardiovasc Res 77: 245–255, 2008.



on May 25, 2011


ownloaded from


156. Haak LL, Song LS, Molinski TF, Pessah IN, Cheng H, Russell

JT. Sparks and puffs in oligodendrocyte progenitors: cross talkbetween ryanodine receptors and inositol trisphosphate receptors.J Neurosci 21: 3860–3870, 2001.

157. Hagiwara S, Nakajima S. Effects of the intracellular Ca ionconcentration upon the excitability of the muscle fiber membraneof a barnacle. J Gen Physiol 49: 807–818, 1966.

158. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Im-proved patch-clamp techniques for high-resolution current record-ing from cells and cell-free membrane patches. Pflugers Arch 391:85–100, 1981.

159. Harkins AB, Kurebayashi N, Baylor SM. Resting myoplasmicfree calcium in frog skeletal muscle fibers estimated with fluo-3.Biophys J 65: 865–881, 1993.

160. Haugland RP. Handbook of Fluorescent Probes and Research

Chemicals. Eugene, OR: Molecular Probes, 2002.161. Hauswirth O, Noble D, Tsien RW. The mechanism of oscillatory

activity at low membrane potentials in cardiac Purkinje fibres.J Physiol 200: 255–265, 1969.

162. Henkart M, Landis DM, Reese TS. Similarity of junctions be-tween plasma membranes and endoplasmic reticulum in muscleand neurons. J Cell Biol 70: 338–347, 1976.

163. Herrera GM, Heppner TJ, Nelson MT. Voltage dependence ofthe coupling of Ca2� sparks to BKCa channels in urinary bladdersmooth muscle. Am J Physiol Cell Physiol 280: C481–C490, 2001.

164. Herrera GM, Nelson MT. Differential regulation of SK and BKchannels by Ca2� signals from Ca2� channels and ryanodine recep-tors in guinea-pig urinary bladder myocytes. J Physiol 541: 483–492, 2002.

165. Hill AP, Sitsapesan R. DIDS modifies the conductance, gating,inactivation mechanisms of the cardiac ryanodine receptor. Bio-

phys J 82: 3037–3047, 2002.166. Hogg RC, Wang Q, Large WA. Time course of spontaneous

calcium-activated chloride currents in smooth muscle cells fromthe rabbit portal vein. J Physiol 464: 15–31, 1993.

167. Hollingworth S, Chandler WK, Baylor SM. Effects of tetracaineon voltage-activated calcium sparks in frog intact skeletal musclefibers. J Gen Physiol 127: 291–307, 2006.

168. Hollingworth S, Peet J, Chandler WK, Baylor SM. Calciumsparks in intact skeletal muscle fibers of the frog. J Gen Physiol

118: 653–678, 2001.169. Huang HP, Wang SR, Yao W, Zhang C, Zhou Y, Chen XW,

Zhang B, Xiong W, Wang LY, Zheng LH, Landry M, Hokfelt T,

Xu ZQD, Zhou Z. Long latency of evoked quantal transmitterrelease from somata of locus coeruleus neurons in rat pontineslices. Proc Natl Acad Sci USA 104: 1401–1406, 2007.

170. Huang LY, Neher E. Ca2�-dependent exocytosis in the somata ofdorsal root ganglion neurons. Neuron 17: 135–145, 1996.

171. Hui CS, Bidasee KR, Besch HR. Effects of ryanodine on calciumsparks in cut twitch fibres of Rana temporaria. J Physiol 534:327–342, 2001.

172. Huser J, Blatter LA. Elementary events of agonist-induced Ca2�

release in vascular endothelial cells. Am J Physiol Cell Physiol 273:C1775–C1782, 1997.

173. Imaizumi Y, Ohi Y, Yamamura H, Ohya S, Muraki K, Watanabe

M. Ca2� spark as a regulator of ion channel activity. Jpn J Phar-

macol 80: 1–8, 1999.174. Inoue M, Bridge JH. Ca2� sparks in rabbit ventricular myocytes

evoked by action potentials involvement of clusters of L-type Ca2�

channels. Circ Res 92: 532–538, 2003.175. Isaeva EV, Shirokova N. Metabolic regulation of Ca2� release in

permeabilized mammalian skeletal muscle fibres. J Physiol 547:453–462, 2003.

176. Isaeva EV, Shkryl VA, Shirokova N. Mitochondrial redox stateand Ca2� sparks in permeabilized mammalian skeletal muscle.J Physiol 565: 855–872, 2005.

177. Ishida H, Genka C, Hirota Y, Nakazawa H, Barry WH. Forma-tion of planar and spiral Ca2� waves in isolated cardiac myocytes.Biophys J 77: 2114–2122, 1999.

178. Izu LT, Banyasz T, Balke CW, Chen-Izu Y. Eavesdropping onthe social lives of Ca2� sparks. Biophys J 93: 3408–3420, 2007.

179. Izu LT, Mauban JR, Balke CW, Wier WG. Large currents gener-ate cardiac Ca2� sparks. Biophys J 80: 88–102, 2001.

180. Izu LT, Means SA, Shadid JN, Chen-Izu Y, Balke CW. Interplayof ryanodine receptor distribution and calcium dynamics. Biophys

J 91: 95–112, 2006.181. Izu LT, Wier WG, Balke CW. Theoretical analysis of the Ca2�

spark amplitude distribution. Biophys J 75: 1144–1162, 1998.182. Janowski E, Cleemann L, Sasse P, Morad M. Diversity of Ca2�

signaling in developing cardiac cells. Ann NY Acad Sci 1080: 154–164, 2006.

183. Ji G, Barsotti RJ, Feldman ME, Kotlikoff MI. Stretch-inducedcalcium release in smooth muscle. J Gen Physiol 119: 533–544,2002.

184. Ji G, Feldman M, Doran R, Zipfel W, Kotlikoff MI. Ca2�-induced Ca2� release through localized Ca2� uncaging in smoothmuscle. J Gen Physiol 127: 225–235, 2006.

185. Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H,

Chen SR. RyR2 mutations linked to ventricular tachycardia andsudden death reduce the threshold for store-overload-inducedCa2� release (SOICR). Proc Natl Acad Sci USA 101: 13062–13067,2004.

186. Jiang YH, Klein MG, Schneider MF. Numerical simulation ofCa2� “sparks” in skeletal muscle. Biophys J 77: 2333–2357, 1999.

187. Jung C, Martins AS, Niggli E, Shirokova N. Dystrophic cardio-myopathy: amplification of cellular damage by Ca2� signalling andreactive oxygen species-generating pathways. Cardiovasc Res 77:766–773, 2008.

188. Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann

DJ, Steinbeck G, McKinnon D, Tomaselli GF. Molecular basisof transient outward potassium current downregulation in humanheart failure: a decrease in Kv4.3 mRNA correlates with a reductionin current density. Circulation 98: 1383–1393, 1998.

189. Kass RS, Lederer WJ, Tsien RW. Current fluctuations in stro-phanthidin-treated cardiac Purkinje fibers (Abstract). Biophys J 16:25a, 1976.

190. Kass RS, Lederer WJ, Tsien RW, Weingart R. Role of calciumions in transient inward currents and aftercontractions induced bystrophanthidin in cardiac Purkinje fibres. J Physiol 281: 187–208,1978.

191. Kass RS, Tsien RW, Weingart R. Ionic basis of transient inwardcurrent induced by strophanthidin in cardiac Purkinje fibres.J Physiol 281: 209–226, 1978.

192. Katoh H, Schlotthauer K, Bers DM. Transmission of informationfrom cardiac dihydropyridine receptor to ryanodine receptor: evi-dence from BayK 8644 effects on resting Ca2� sparks. Circ Res 87:106–111, 2000.

193. Keizer J, Smith GD, Ponce-Dawson S, Pearson JE. Saltatorypropagation of Ca2� waves by Ca2� sparks. Biophys J 75: 595–600,1998.

194. Kettlun C, Gonzalez A, Rios E, Fill M. Unitary Ca2� currentthrough mammalian cardiac and amphibian skeletal muscleryanodine receptor channels under near-physiological ionic condi-tions. J Gen Physiol 122: 407–417, 2003.

195. Kim M, Han IS, Koh SD, Perrino BA. Roles of CaM kinase II andphospholamban in SNP-induced relaxation of murine gastric fun-dus smooth muscles. Am J Physiol Cell Physiol 291: C337–C347,2006.

196. Kirchhof P, Klimas J, Fabritz L, Zwiener M, Jones LR,

Schafers M, Hermann S, Boknik P, Schmitz W, Breithardt G,

Kirchhefer U, Neumann J. Stress and high heart rate provokeventricular tachycardia in mice expressing triadin. J Mol Cell Car-

diol 42: 962–971, 2007.197. Klein MG, Cheng H, Santana LF, Jiang YH, Lederer WJ,

Schneider MF. Two mechanisms of quantized calcium release inskeletal muscle. Nature 379: 455–458, 1996.

198. Klein MG, Lacampagne A, Schneider MF. A repetitive mode ofactivation of discrete Ca2� release events (Ca2� sparks) in frogskeletal muscle fibres. J Physiol 515: 391–411, 1999.

199. Klein MG, Lacampagne A, Schneider MF. Voltage dependenceof the pattern and frequency of discrete Ca2� release events afterbrief repriming in frog skeletal muscle. Proc Natl Acad Sci USA 94:11061–11066, 1997.

200. Klein MG, Schneider MF. Ca2� sparks in skeletal muscle. Prog

Biophys Mol Biol 92: 308–332, 2006.

CALCIUM SPARKS 1539


on May 25, 2011


ownloaded from


201. Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn

K, Knollmann BE, Horton KD, Weissman NJ, Holinstat I,

Zhang W, Roden DM, Jones LR, Franzini-Armstrong C, Pfeifer

K. Casq2 deletion causes sarcoplasmic reticulum volume increase,premature Ca2� release, catecholaminergic polymorphic ventricu-lar tachycardia. J Clin Invest 116: 2510–2520, 2006.

202. Koizumi S, Bootman MD, Bobanovic LK, Schell MJ, Berridge

MJ, Lipp P. Characterization of elementary Ca2� release signals inNGF-differentiated PC12 cells and hippocampal neurons. Neuron

22: 125–137, 1999.203. Kotlikoff MI, Wang YX. Calcium release and calcium-activated

chloride channels in airway smooth muscle cells. Am J Respir Crit

Care Med 158: S109–S114, 1998.204. Kovacs L, Rios E, Schneider MF. Calcium transients and in-

tramembrane charge movement in skeletal muscle fibres. Nature

279: 391–396, 1979.205. Lacampagne A, Klein MG, Schneider MF. Modulation of the

frequency of spontaneous sarcoplasmic reticulum Ca2� releaseevents (Ca2� sparks) by myoplasmic [Mg2�] in frog skeletal mus-cle. J Gen Physiol 111: 207–224, 1998.

206. Lacampagne A, Klein MG, Ward CW, Schneider MF. Twomechanisms for termination of individual Ca2� sparks in skeletalmuscle. Proc Natl Acad Sci USA 97: 7823–7828, 2000.

207. Lacombe VA, Viatchenko-Karpinski S, Terentyev D, Sridhar

A, Emani S, Bonagura JD, Feldman DS, Gyorke S, Carnes CA.

Mechanisms of impaired calcium handling underlying subclinicaldiastolic dysfunction in diabetes. Am J Physiol Regul Integr Comp

Physiol 293: R1787–R1797, 2007.208. Lakatta EG, Vinogradova T, Lyashkov A, Sirenko S, Zhu W,

Ruknudin A, Maltsev VA. The integration of spontaneous intra-cellular Ca2� cycling and surface membrane ion channel activationentrains normal automaticity in cells of the heart’s pacemaker. Ann

NY Acad Sci 1080: 178–206, 2006.209. Lamb GD. Ryanodine receptor “adaptation”: a flash in the pan?

J Muscle Res Cell Motil 18: 611–616, 1997.210. Lamb GD, Laver DR, Stephenson DG. Questions about adapta-

tion in ryanodine receptors. J Gen Physiol 116: 883–890, 2000.211. Lamb GD, Stephenson DG. Activation of ryanodine receptors by

flash photolysis of caged Ca2�. Biophys J 68: 946–948, 1995.212. Langer GA, Peskoff A. Calcium concentration and movement in

the diadic cleft space of the cardiac ventricular cell. Biophys J 70:1169–1182, 1996.

213. Launikonis BS, Zhou J, Royer L, Shannon TR, Brum G, Rios E.

Depletion “skraps” and dynamic buffering inside the cellular cal-cium store. Proc Natl Acad Sci USA 103: 2982–2987, 2006.

214. Launikonis BS, Zhou J, Santiago D, Brum G, Rios E. Thechanges in Ca2� sparks associated with measured modifications ofintra-store Ca2� concentration in skeletal muscle. J Gen Physiol

128: 45–54, 2006.215. Laver DR, Lamb GD. Inactivation of Ca2� release channels

(ryanodine receptors RyR1 and RyR2) with rapid steps in [Ca2�]and voltage. Biophys J 74: 2352–2364, 1998.

216. Lebeche D, Kaprielian R, Hajjar R. Modulation of action poten-tial duration on myocyte hypertrophic pathways. J Mol Cell Cardiol

40: 725–735, 2006.217. Lechleiter J, Girard S, Peralta E, Clapham D. Spiral calcium

wave propagation and annihilation in Xenopus laevis oocytes.Science 252: 123–126, 1991.

218. Lechleiter JD, Clapham DE. Molecular mechanisms of intracel-lular calcium excitability in X. laevis oocytes. Cell 69: 283–294,1992.

219. Lederer WJ, Tsien RW. Transient inward current underlyingarrhythmogenic effects of cardiotonic steroids in Purkinje fibres.J Physiol 263: 73–100, 1976.

220. Lehnart SE, Terrenoire C, Reiken S, Wehrens XH, Song LS,

Tillman EJ, Mancarella S, Coromilas J, Lederer WJ, Kass RS,

Marks AR. Stabilization of cardiac ryanodine receptor preventsintracellular calcium leak and arrhythmias. Proc Natl Acad Sci USA

103: 7906–7910, 2006.221. Lehnart SE, Wehrens XH, Laitinen PJ, Reiken SR, Deng SX,

Cheng Z, Landry DW, Kontula K, Swan H, Marks AR. Suddendeath in familial polymorphic ventricular tachycardia associated

with calcium release channel (ryanodine receptor) leak. Circula-

tion 109: 3208–3214, 2004.222. Lehnart SE, Wehrens XH, Reiken S, Warrier S, Belevych AE,

Harvey RD, Richter W, Jin SL, Conti M, Marks AR. Phospho-diesterase 4D deficiency in the ryanodine-receptor complex pro-motes heart failure and arrhythmias. Cell 123: 25–35, 2005.

223. Lesh RE, Nixon GF, Fleischer S, Airey JA, Somlyo AP, Somlyo

AV. Localization of ryanodine receptors in smooth muscle. Circ

Res 82: 175–185, 1998.224. Li Y, Bers DM. A cardiac dihydropyridine receptor II–III loop

peptide inhibits resting Ca2� sparks in ferret ventricular myocytes.J Physiol 537: 17–26, 2001.

225. Li Y, Kranias EG, Mignery GA, Bers DM. Protein kinase Aphosphorylation of the ryanodine receptor does not affect calciumsparks in mouse ventricular myocytes. Circ Res 90: 309–316, 2002.

226. Lindner M, Brandt MC, Sauer H, Hescheler J, Bohle T, Beuck-

elmann DJ. Calcium sparks in human ventricular cardiomyocytesfrom patients with terminal heart failure. Cell Calcium 31: 175–182,2002.

227. Liou J, Kim ML, Heo WD, Jones JT, Myers JW, Ferrell JE,

Meyer T. STIM is a Ca2� sensor essential for Ca2�-store-depletion-triggered Ca2� influx. Curr Biol 15: 1235–1241, 2005.

228. Lipp P, Egger M, Niggli E. Spatial characteristics of sarcoplasmicreticulum Ca2� release events triggered by L-type Ca2� current andNa� current in guinea-pig cardiac myocytes. J Physiol 542: 383–393, 2002.

229. Lipp P, Laine M, Tovey SC, Burrell KM, Berridge MJ, Li W,

Bootman MD. Functional InsP3 receptors that may modulateexcitation-contraction coupling in the heart. Curr Biol 10: 939–942,2000.

230. Lipp P, Niggli E. Fundamental calcium release events revealed bytwo-photon excitation photolysis of caged calcium in guinea-pigcardiac myocytes. J Physiol 508: 801–809, 1998.

231. Lipp P, Niggli E. Microscopic spiral waves reveal positive feed-back in subcellular calcium signaling. Biophys J 65: 2272–2276,1993.

232. Lipp P, Niggli E. Submicroscopic calcium signals as fundamentalevents of excitation–contraction coupling in guinea-pig cardiacmyocytes. J Physiol 492: 31–38, 1996.

233. Litwin SE, Zhang D, Bridge JH. Dyssynchronous Ca2� sparks inmyocytes from infarcted hearts. Circ Res 87: 1040–1047, 2000.

234. Liu N, Priori SG. Disruption of calcium homeostasis and arrhyth-mogenesis induced by mutations in the cardiac ryanodine receptorand calsequestrin. Cardiovasc Res 77: 293–301, 2008.

235. Liu QH, Zheng YM, Wang YX. Two distinct signaling pathways forregulation of spontaneous local Ca2� release by phospholipase C inairway smooth muscle cells. Pflugers Arch 453: 531–541, 2007.

236. Llano I, Gonzalez J, Caputo C, Lai FA, Blayney LM, Tan YP,

Marty A. Presynaptic calcium stores underlie large-amplitude min-iature IPSCs and spontaneous calcium transients. Nat Neurosci 3:1256–1265, 2000.

237. Loesser KE, Castellani L, Franzini-Armstrong C. Dispositionsof junctional feet in muscles of invertebrates. J Muscle Res Cell

Motil 13: 161–173, 1992.238. Lokuta AJ, Komai H, McDowell TS, Valdivia HH. Functional

properties of ryanodine receptors from rat dorsal root ganglia.FEBS Lett 511: 90–96, 2002.

239. Lopez-Lopez JR, Shacklock PS, Balke CW, Wier WG. Local,stochastic release of Ca2� in voltage-clamped rat heart cells: visu-alization with confocal microscopy. J Physiol 480: 21–29, 1994.

240. Lopez-Lopez JR, Shacklock PS, Balke CW, Wier WG. Localcalcium transients triggered by single L-type calcium channel cur-rents in cardiac cells. Science 268: 1042–1045, 1995.

241. Louch WE, Bito V, Heinzel FR, Macianskiene R, Vanhaecke J,

Flameng W, Mubagwa K, Sipido KR. Reduced synchrony of Ca2�

release with loss of T-tubules—a comparison to Ca2� release inhuman failing cardiomyocytes. Cardiovasc Res 62: 63–73, 2004.

242. Lukyanenko V, Gyorke S. Ca2� sparks and Ca2� waves in sapo-nin-permeabilized rat ventricular myocytes. J Physiol 521: 575–585,1999.

243. Lukyanenko V, Subramanian S, Gyorke I, Wiesner TF, Gyorke

S. The role of luminal Ca2� in the generation of Ca2� waves in ratventricular myocytes. J Physiol 518: 173–186, 1999.



on May 25, 2011


ownloaded from


244. Luo D, Yang D, Lan X, Li K, Li X, Chen J, Zhang Y, Xiao RP,

Han Q, Cheng H. Nuclear Ca2� sparks and waves mediated byinositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyo-cytes. Cell Calcium 2007.

245. Macrez N, Mironneau J. Local Ca2� signals in cellular signalling.Curr Mol Med 4: 263–275, 2004.

246. Mak DD, McBride S, Foskett JK. Inositol 1,4,5-tris-phosphateactivation of inositol tris-phosphate receptor Ca2� channel by li-gand tuning of Ca2� inhibition. Proc Natl Acad Sci USA 95: 15821–15825, 1998.

247. Marchant JS, Parker I. Role of elementary Ca2� puffs in gener-ating repetitive Ca2� oscillations. EMBO J 20: 65–76, 2001.

248. Marks AR, Priori S, Memmi M, Kontula K, Laitinen PJ. In-volvement of the cardiac ryanodine receptor/calcium release chan-nel in catecholaminergic polymorphic ventricular tachycardia.J Cell Physiol 190: 1–6, 2002.

249. Marx SO, Gaburjakova J, Gaburjakova M, Henrikson C,

Ondrias K, Marks AR. Coupled gating between cardiac calciumrelease channels (ryanodine receptors). Circ Res 88: 1151–1158,2001.

250. Marx SO, Kurokawa J, Reiken S, Motoike H, D’Armiento J,

Marks AR, Kass RS. Requirement of a macromolecular signalingcomplex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 295: 496–499, 2002.

251. Marx SO, Ondrias K, Marks AR. Coupled gating between indi-vidual skeletal muscle Ca2� release channels (ryanodine recep-tors). Science 281: 818–821, 1998.

252. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D,

Rosemblit N, Marks AR. PKA phosphorylation dissociatesFKBP12.6 from the calcium release channel (ryanodine receptor):defective regulation in failing hearts. Cell 101: 365–376, 2000.

253. Mauban JR, Lamont C, Balke CW, Wier WG. Adrenergic stim-ulation of rat resistance arteries affects Ca2� sparks, Ca2� waves,Ca2� oscillations. Am J Physiol Heart Circ Physiol 280: H2399–H2405, 2001.

254. Meethal SV, Potter KT, Redon D, Munoz-Del-Rio A, Kamp TJ,

Valdivia HH, Haworth RA. Structure-function relationships ofCa2� spark activity in normal and failing cardiac myocytes asrevealed by flash photography. Cell Calcium 41: 123–134, 2007.

255. Meissner G. Ryanodine receptor/Ca2� release channels and theirregulation by endogenous effectors. Annu Rev Physiol 56: 485–508,1994.

256. Meissner G. Molecular regulation of cardiac ryanodine receptorion channel. Cell Calcium 35: 621–628, 2004.

257. Melamed-Book N, Kachalsky SG, Kaiserman I, Rahamimoff R.

Neuronal calcium sparks and intracellular calcium “noise.” Proc

Natl Acad Sci USA 96: 15217–15221, 1999.258. Melzer W. When sparks get old. J Cell Biol 174: 613–614, 2006.259. Meyers MB, Fischer A, Sun YJ, Lopes CM, Rohacs T,

Nakamura TY, Zhou YY, Lee PC, Altschuld RA, McCune SA,

Coetzee WA, Fishman GI. Sorcin regulates excitation-contrac-tion coupling in the heart. J Biol Chem 278: 28865–28871, 2003.

260. Miake J, Marban E, Nuss HB. Functional role of inward rectifiercurrent in heart probed by Kir2.1 overexpression and dominant-negative suppression. J Clin Invest 111: 1529–1536, 2003.

261. Minta A, Kao JP, Tsien RY. Fluorescent indicators for cytosoliccalcium based on rhodamine and fluorescein chromophores. J Biol

Chem 264: 8171–8178, 1989.262. Miriel VA, Mauban JR, Blaustein MP, Wier WG. Local and

cellular Ca2� transients in smooth muscle of pressurized rat resis-tance arteries during myogenic and agonist stimulation. J Physiol

518: 815–824, 1999.263. Mironneau J, Arnaudeau S, Macrez-Lepretre N, Boittin FX.

Ca2� sparks and Ca2� waves activate different Ca2�-dependent ionchannels in single myocytes from rat portal vein. Cell Calcium 20:153–160, 1996.

264. Missiaen L, Taylor CW, Berridge MJ. Spontaneous calciumrelease from inositol trisphosphate-sensitive calcium stores.Nature 352: 241–244, 1991.

265. Mori MX, Erickson MG, Yue DT. Functional stoichiometry andlocal enrichment of calmodulin interacting with Ca2� channels.Science 304: 432–435, 2004.

266. Morimura K, Ohi Y, Yamamura H, Ohya S, Muraki K, Imaizumi

Y. Two-step Ca2� intracellular release underlies excitation-contrac-tion coupling in mouse urinary bladder myocytes. Am J Physiol

Cell Physiol 290: C388–C403, 2006.267. Moschella MC, Marks AR. Inositol 1,4,5-trisphosphate receptor

expression in cardiac myocytes. J Cell Biol 120: 1137–1146, 1993.268. Mullins LJ. The role of Na-Ca exchange in heart. In: Physiology

and Pathophysiology of the Heart, edited by Sperelakis N. Lancaster,PA: Martinus Nijhoff, 1984, p. 199–214.

269. Nabauer M, Kaab S. Potassium channel down-regulation in heartfailure. Cardiovasc Res 37: 324–334, 1998.

270. Nabauer M, Morad M. Ca2�-induced Ca2� release as examined byphotolysis of caged Ca2� in single ventricular myocytes. Am J

Physiol Cell Physiol 258: C189–C193, 1990.271. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA,

Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apop-tosis and cytotoxicity by amyloid-beta. Nature 403: 98–103, 2000.

272. Navedo MF, Amberg GC, Nieves M, Molkentin JD, Santana

LF. Mechanisms underlying heterogeneous Ca2� sparklet activityin arterial smooth muscle. J Gen Physiol 127: 611–622, 2006.

273. Navedo MF, Amberg GC, Votaw VS, Santana LF. Constitutivelyactive L-type Ca2� channels. Proc Natl Acad Sci USA 102: 11112–11117, 2005.

274. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot

HJ, Lederer WJ. Relaxation of arterial smooth muscle by calciumsparks. Science 270: 633–637, 1995.

275. Nerbonne JM, Kass RS. Molecular physiology of cardiac repolar-ization. Physiol Rev 85: 1205–1253, 2005.

276. Niggli E. The cardiac sarcoplasmic reticulum: filled with Ca2� andsurprises. Circ Res 100: 5–6, 2007.

277. Niggli E. Localized intracellular calcium signaling in muscle: cal-cium sparks and calcium quarks. Annu Rev Physiol 61: 311–335,1999.

278. Niggli E, Egger M. Calcium quarks. Front Biosci 7: d1288–d1297,2002.

279. Niggli E, Lederer WJ. Voltage-independent calcium release inheart muscle. Science 250: 565–568, 1990.

280. Niggli E, Shirokova N. A guide to sparkology: the taxonomy ofelementary cellular Ca2� signaling events. Cell Calcium 42: 379–387, 2007.

281. Nuss HB, Kaab S, Kass DA, Tomaselli GF, Marban E. Cellularbasis of ventricular arrhythmias and abnormal automaticity inheart failure. Am J Physiol Heart Circ Physiol 277: H80–H91, 1999.

282. Nuss HB, Marban E, Johns DC. Overexpression of a humanpotassium channel suppresses cardiac hyperexcitability in rabbitventricular myocytes. J Clin Invest 103: 889–896, 1999.

283. Ouyang K, Wu C, Cheng H. Ca2�-induced Ca2� release in sensoryneurons: low gain amplification confers intrinsic stability. J Biol

Chem 280: 15898–15902, 2005.284. Ouyang K, Zheng H, Qin X, Zhang C, Yang D, Wang X, Wu C,

Zhou Z, Cheng H. Ca2� sparks and secretion in dorsal root gan-glion neurons. Proc Natl Acad Sci USA 102: 12259–12264, 2005.

285. Pacher P, Thomas AP, Hajnoczky G. Ca2� marks: miniaturecalcium signals in single mitochondria driven by ryanodine recep-tors. Proc Natl Acad Sci USA 99: 2380–2385, 2002.

286. Pape PC, Jong DS, Chandler WK, Baylor SM. Effect of fura-2 onaction-potential stimulated calcium-release in cut twitch fibersfrom frog-muscle. J Gen Physiol 102: 295–332, 1993.

287a.Parker I, Choi J, Yao Y. Elementary events of InsP3-induced Ca2�

liberation in Xenopus oocytes: hot spots, puffs and blips. Cell

Calcium 20: 105–121, 1996.288. Parker I, Wier WG. Variability in frequency and characteristics of

Ca2� sparks at different release sites in rat ventricular myocytes.J Physiol 505: 337–344, 1997.

289. Parker I, Yao Y. Relation between intracellular Ca2� signals andCa2�- activated Cl� current in Xenopus oocytes. Cell Calcium 15:276–288, 1994.

290. Peng S, Publicover NG, Kargacin GJ, Duan D, Airey JA, Sutko

JL. Imaging single cardiac ryanodine receptor Ca2� fluxes in lipidbilayers. Biophys J 86: 134–144, 2004.

291. Pereira L, Matthes J, Schuster I, Valdivia HH, Herzig S, Richard

S, Gomez AM. Mechanisms of [Ca2�]i transient decrease in cardio-

CALCIUM SPARKS 1541


on May 25, 2011


ownloaded from


myopathy of db/db type 2 diabetic mice. Diabetes 55: 608–615,2006.

292. Perez GJ, Bonev AD, Nelson MT. Micromolar Ca2� from sparksactivates Ca2�-sensitive K� channels in rat cerebral artery smoothmuscle. Am J Physiol Cell Physiol 281: C1769–C1775, 2001.

293. Perez GJ, Bonev AD, Patlak JB, Nelson MT. Functional cou-pling of ryanodine receptors to K-Ca channels in smooth musclecells from rat cerebral arteries. J Gen Physiol 113: 229–237, 1999.

294. Petroff MGV, Kim SH, Pepe S, Dessy C, Marban E, Balligand

JL, Sollott SJ. Endogenous nitric oxide mechanisms mediate thestretch dependence of Ca2� release in cardiomyocytes. Nat Cell

Biol 3: 867–873, 2001.295. Picht E, Zima AV, Blatter LA, Bers DM. SparkMaster: automated

calcium spark analysis with ImageJ. Am J Physiol Cell Physiol 293:C1073–C1081, 2007.

296. Pizarro G, Shirokova N, Tsugorka A, Rios E. “Quantal” calciumrelease operated by membrane voltage in frog skeletal muscle.J Physiol 501: 289–303, 1997.

297. Pluger S, Faulhaber J, Furstenau M, Lohn M, Waldschutz R,

Gollasch M, Haller H, Luft FC, Ehmke H, Pongs O. Mice withdisrupted BK channel beta1 subunit gene feature abnormal Ca2�

spark/STOC coupling and elevated blood pressure. Circ Res 87:E53–E60, 2000.

298. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhyth-mogenesis and contractile dysfunction in heart failure: roles ofsodium-calcium exchange, inward rectifier potassium current, re-sidual beta-adrenergic responsiveness. Circ Res 88: 1159–1167,2001.

299. Poindexter BJ, Smith JR, Buja LM, Bick RJ. Calcium signalingmechanisms in dedifferentiated cardiac myocytes: comparisonwith neonatal and adult cardiomyocytes. Cell Calcium 30: 373–382,2001.

300. Pouvreau S, Royer L, Yi JX, Brum G, Meissner G, Rios E, Zhou

JS. Ca2� sparks operated by membrane depolarization requireisoform 3 ryanodine receptor channels in skeletal muscle. Proc

Natl Acad Sci USA 104: 5235–5240, 2007.301. Pratusevich VR, Balke CW. Factors shaping the confocal image

of the calcium spark in cardiac muscle cells. Biophys J 71: 2942–2957, 1996.

302. Pucovsky V, Gordienko DV, Bolton TB. Effect of nitric oxidedonors and noradrenaline on Ca2� release sites and global intra-cellular Ca2� in myocytes from guinea-pig small mesenteric arter-ies. J Physiol 539: 25–39, 2002.

303. Putney JW Jr, Broad LM, Braun FJ, Lievremont JP, Bird GS.

Mechanisms of capacitative calcium entry. J Cell Sci 114: 2223–2229, 2001.

304. Rengifo J, Rosales R, Gonzalez A, Cheng H, Stern MD, Rios E.

Intracellular Ca2� release as irreversible Markov process. Biophys

J 83: 2511–2521, 2002.305. Ridgway EB, Gilkey JC, Jaffe LF. Free calcium increases explo-

sively in activating medaka eggs. Proc Natl Acad Sci USA 74:623–627, 1977.

306. Rios E, Brum G. Ca2� release flux underlying Ca2� transients andCa2� sparks in skeletal muscle. Front Biosci 7: d1195–d1211, 2002.

307. Rios E, Pizarro G. Voltage sensor of excitation-contraction cou-pling in skeletal muscle. Physiol Rev 71: 849–908, 1991.

308. Rios E, Shirokova N, Kirsch WG, Pizarro G, Stern MD, Cheng

H, Gonzalez A. A preferred amplitude of calcium sparks in skel-etal muscle. Biophys J 80: 169–183, 2001.

309. Rizzuto R, Pozzan T. Microdomains of intracellular Ca2�: molec-ular determinants and functional consequences. Physiol Rev 86:369–408, 2006.

310. Rose HJ, Dargan S, Shuai JW, Parker I. “Trigger” events pre-cede calcium puffs in Xenopus oocytes. Biophys J 91: 4024–4032,2006.

311. Rosenbluth J. Subsurface cisterns and their relationship to theneuronal plasma membrane. J Cell Biol 13: 405–421, 1962.

312. Rossow CF, Minami E, Chase EG, Murry CE, Santana LF.

NFATc3-induced reductions in voltage-gated K� currents aftermyocardial infarction. Circ Res 94: 1340–1350, 2004.

313. Sah R, Ramirez RJ, Kaprielian R, Backx PH. Alterations inaction potential profile enhance excitation-contraction coupling inrat cardiac myocytes. J Physiol 533: 201–214, 2001.

314. Sah R, Ramirez RJ, Backx PH. Modulation of Ca2� release incardiac myocytes by changes in repolarization rate: role of phase-1action potential repolarization in excitation-contraction coupling.Circ Res 90: 165–173, 2002.

315. Santana LF, Cheng H, Gomez AM, Cannell MB, Lederer WJ.

Relation between the sarcolemmal Ca2� current and Ca2� sparksand local control theories for cardiac excitation-contraction cou-pling. Circ Res 78: 166–171, 1996.

316. Satoh H, Blatter LA, Bers DM. Effects of [Ca2�]i, SR Ca2� load,rest on Ca2� spark frequency in ventricular myocytes. Am J

Physiol Heart Circ Physiol 272: H657–H668, 1997.317. Satoh H, Delbridge LM, Blatter LA, Bers DM. Surface:volume

relationship in cardiac myocytes studied with confocal microscopyand membrane capacitance measurements: species-dependenceand developmental effects. Biophys J 70: 1494–1504, 1996.

318. Satoh H, Hayashi H, Blatter LA, Bers DM. BayK 8644 increasesresting calcium spark frequency in ferret ventricular myocytes.Heart Vessels Suppl 12: 58–61, 1997.

319. Satoh H, Katoh H, Velez P, Fill M, Bers DM. Bay K 8644increases resting Ca2� spark frequency in ferret ventricular myo-cytes independent of Ca influx: contrast with caffeine and ryano-dine effects. Circ Res 83: 1192–1204, 1998.

320. Schneider MF, Chandler WK. Voltage dependent charge move-ment in skeletal-muscle: possible step in excitation-contractioncoupling. Nature 242: 244–246, 1973.

321. Schneider MF. Ca2� sparks in frog skeletal muscle: generation byone, some, or many SR Ca2� release channels? J Gen Physiol 113:365–372, 1999.

322. Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli

MD, Pozzan T, Korsmeyer SJ. BAX and BAK regulation of en-doplasmic reticulum Ca2�: a control point for apoptosis. Science

300: 135–139, 2003.323. Sham JS, Cleemann L, Morad M. Functional coupling of Ca2�

channels and ryanodine receptors in cardiac myocytes. Proc Natl

Acad Sci USA 92: 121–125, 1995.324. Sham JS, Song LS, Chen Y, Deng LH, Stern MD, Lakatta EG,

Cheng H. Termination of Ca2� release by a local inactivation ofryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA

95: 15096–15101, 1998.325. Sham JSK, Cleemann L, Morad M. Gating of the cardiac Ca2�

release channel: the role of Na� current and Na�-Ca2� exchange.Science 255: 850–853, 1992.

326. Shannon TR. Linking calsequestrin to lumenal control of SR Ca2�

release. Circ Res 101: 539–541, 2007.327. Shannon TR, Guo T, Bers DM. Ca2� scraps: local depletions of

free [Ca2�] in cardiac sarcoplasmic reticulum during contractionsleave substantial Ca2� reserve. Circ Res 93: 40–45, 2003.

328. Shannon TR, Wang F, Bers DM. Regulation of cardiac sarcoplas-mic reticulum Ca2� release by luminal Ca2� and altered gatingassessed with a mathematical model. Biophys J 89: 4096–4110,2005.

329. Shao CH, Rozanski GJ, Patel KP, Bidasee KR. Dyssynchronous(non-uniform) Ca2� release in myocytes from streptozotocin-in-duced diabetic rats. J Mol Cell Cardiol 42: 234–246, 2007.

330. Sheehan KA, Zima AV, Blatter LA. Regional differences in spon-taneous Ca spark activity and regulation in cat atrial myocytes.J Physiol 572: 799–809, 2006.

331. Shen JX, Wang S, Song LS, Han T, Cheng H. Polymorphism ofCa2� sparks evoked from in-focus Ca2� release units in cardiacmyocytes. Biophys J 86: 182–190, 2004.

332. Shirokova N, Garcia J, Rios E. Local calcium release in mam-malian skeletal muscle. J Physiol 512: 377–384, 1998.

333. Shirokova N, Garcia J, Pizarro G, Rios E. Ca2� release from thesarcoplasmic reticulum compared in amphibian and mammalianskeletal muscle. J Gen Physiol 107: 1–18, 1996.

334. Shirokova N, Gonzalez A, Kirsch WG, Rios E, Pizarro G, Stern

MD, Cheng H. Calcium sparks: release packets of uncertain originand fundamental role. J Gen Physiol 113: 377–384, 1999.

335. Shirokova N, Rios E. Small event Ca2� release: a probable pre-cursor of Ca2� sparks in frog skeletal muscle. J Physiol 502: 3–11,1997.

336. Shirokova N, Shirokov R, Rossi D, Gonzalez A, Kirsch WG,

Garcia J, Sorrentino V, Rios E. Spatially segregated control of



on May 25, 2011


ownloaded from


Ca2� release in developing skeletal muscle of mice. J Physiol 521:483–495, 1999.

337. Shkryl VM, Shirokova N. Transfer and tunneling of Ca2� fromsarcoplasmic reticulum to mitochondria in skeletal muscle. J Biol

Chem 281: 1547–1554, 2006.338. Shuai JW, Rose HJ, Parker I. The number and spatial distribu-

tion of IP3 receptors underlying calcium puffs in Xenopus oocytes.Biophys J 91: 4033–4044, 2006.

339. Sieck GC, Kannan MS, Prakash YS. Heterogeneity in dynamicregulation of intracellular calcium in airway smooth muscle cells.Can J Physiol Pharmacol 75: 878–888, 1997.

340. Simon BJ, Klein MG, Schneider MF. Calcium dependence ofinactivation of calcium release from the sarcoplasmic-reticulum inskeletal-muscle fibers. J Gen Physiol 97: 437–471, 1991.

341. Singh J, Chonkar A, Bracken N, Adeghate E, Latt Z, Hussain

M. Effect of streptozotocin-induced type 1 diabetes mellitus oncontraction, calcium transient, cation contents in the isolated ratheart. Ann NY Acad Sci 1084: 178–190, 2006.

342. Sipido KR, Volders PG, Vos MA, Verdonck F. Altered Na/Caexchange activity in cardiac hypertrophy and heart failure: a newtarget for therapy? Cardiovasc Res 53: 782–805, 2002.

343. Sitsapesan R, Williams AJ. Do inactivation mechanisms ratherthan adaptation hold the key to understanding ryanodine receptorchannel gating? J Gen Physiol 116: 867–872, 2000.

344. Smith GD, Keizer JE, Stern MD, Lederer WJ, Cheng H. Asimple numerical model of calcium spark formation and detectionin cardiac myocytes. Biophys J 75: 15–32, 1998.

345. Sobie EA, Cannell MB, Bridge JH. Allosteric activation of Na�-Ca2� exchange by L-type Ca2� current augments the trigger flux forSR Ca2� release in ventricular myocytes. Biophys J 94: 54–56,2008.

346. Sobie EA, Dilly KW, dos Santos CJ, Lederer WJ, Jafri MS.

Termination of cardiac Ca2� sparks: an investigative mathematicalmodel of calcium-induced calcium release. Biophys J 83: 59–78,2002.

347. Sobie EA, Guatimosim S, Gomez-Viquez L, Song LS, Hart-

mann H, Saleet JM, Lederer WJ. The Ca2� leak paradox and“rogue ryanodine receptors”: SR Ca2� efflux theory and practice.Prog Biophys Mol Biol 90: 172–185, 2006.

349. Sobie EA, Song LS, Lederer WJ. Local recovery of Ca2� releasein rat ventricular myocytes. J Physiol 565: 441–447, 2005.

350. Soeller C, Cannell MB. Numerical simulation of local calciummovements during L-type calcium channel gating in the cardiacdiad. Biophys J 73: 97–111, 1997.

351. Soeller C, Cannell MB. Estimation of the sarcoplasmic reticulumCa2� release flux underlying Ca2� sparks. Biophys J 82: 2396–2414,2002.

352. Soeller C, Crossman D, Gilbert R, Cannell MB. Analysis ofryanodine receptor clusters in rat and human cardiac myocytes.Proc Natl Acad Sci USA 104: 14958–14963, 2007.

353. Song L, Alcalai R, Arad M, Wolf CM, Toka O, Conner DA,

Berul CI, Eldar M, Seidman CE, Seidman JG. Calsequestrin 2(CASQ2) mutations increase expression of calreticulin and ryano-dine receptors, causing catecholaminergic polymorphic ventriculartachycardia. J Clin Invest 117: 1814–1823, 2007.

354. Song LS, Guatimosim S, Gomez-Viquez L, Sobie EA, Ziman A,

Hartmann H, Lederer WJ. Calcium biology of the transversetubules in heart. Ann NY Acad Sci 1047: 99–111, 2005.

355. Song LS, Pi Y, Kim SJ, Yatani A, Guatimosim S, Kudej RK,

Zhang Q, Cheng H, Hittinger L, Ghaleh B, Vatner DE, Lederer

WJ, Vatner SF. Paradoxical cellular Ca2� signaling in severe butcompensated canine left ventricular hypertrophy. Circ Res 97:457–464, 2005.

356. Song LS, Pi YQ, Guatimosim S, Kim SJ, Yatani A, Kudej RK,

Zhang QX, Hittinger L, Ghaleh B, Cheng HP, Vatner DE,

Lederer WJ, Vatner SF. Impaired cellular Ca2� handling in com-pensated canine left ventricular hypertrophy (Abstract). Biophys J

88: 135A–136A, 2005.357. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. Direct

measurement of SR release flux by tracking “Ca2� spikes” in ratcardiac myocytes. J Physiol 512: 677–691, 1998.

358. Song LS, Sobie EA, McCulle S, Lederer WJ, Balke CW, Cheng

H. Orphaned ryanodine receptors in the failing heart. Proc Natl

Acad Sci USA 103: 4305–4310, 2006.359. Song LS, Stern MD, Lakatta EG, Cheng H. Partial depletion of

sarcoplasmic reticulum calcium does not prevent calcium sparks inrat ventricular myocytes. J Physiol 505: 665–675, 1997.

360. Song LS, Wang SQ, Xiao RP, Spurgeon H, Lakatta EG, Cheng

H. �-Adrenergic stimulation synchronizes intracellular Ca2� re-lease during excitation-contraction coupling in cardiac myocytes.Circ Res 88: 794–801, 2001.

361. Song LS, Sobie EA, McCulle S, Lederer WJ, Balke WC, Cheng

H. Orphaned ryanodine receptors in the failing heart. Proc Natl

Acad Sci USA 103: 4305–4310, 2006.362. Stern MD. Buffering of calcium in the vicinity of a channel pore.

Cell Calcium 13: 183–192, 1992.363. Stern MD. Theory of excitation-contraction coupling in cardiac

muscle. Biophys J 63: 497–517, 1992.364. Stern MD, Cheng H. Putting out the fire: what terminates calcium-

induced calcium release in cardiac muscle? Cell Calcium 35: 591–601, 2004.

365. Stern MD, Kort AA, Bhatnagar GM, Lakatta EG. Scattered-light intensity fluctuations in diastolic rat cardiac-muscle caused byspontaneous Ca2�-dependent cellular mechanical oscillations.J Gen Physiol 82: 119–153, 1983.

366. Stern MD, Pizarro G, Rios E. Local control model of excitation-contraction coupling in skeletal muscle. J Gen Physiol 110: 415–440, 1997.

367. Stern MD, Song LS, Cheng H, Sham JS, Yang HT, Boheler KR,

Rios E. Local control models of cardiac excitation-contractioncoupling. A possible role for allosteric interactions betweenryanodine receptors. J Gen Physiol 113: 469–489, 1999.

368. Stuyvers BD, Dun W, Matkovich S, Sorrentino V, Boyden PA,

ter Keurs HEDJ Ca2� sparks and waves in canine Purkinje cells:a triple layered system of Ca2� activation. Circ Res 97: 35–43, 2005.

369. Sun XP, Callamaras N, Marchant JS, Parker I. A continuum ofInsP3-mediated elementary Ca2� signalling events in Xenopus

oocytes. J Physiol 509: 67–80, 1998.370. Tan WC, Fu CQ, Fu CJ, Xie WJ, Cheng HP. An anomalous

subdiffusion model for calcium spark in cardiac myocytes. Appl

Phys Lett 91: 2007.371. Tanaka H, Sekine T, Kawanishi T, Nakamura R, Shigenobu K.

Intrasarcomere Ca2� gradients and their spatio-temporal relationto Ca2� sparks in rat cardiomyocytes. J Physiol 508: 145–152, 1998.

372. Ter Keurs HEDJ, Boyden PA. Calcium and arrhythmogenesis.Physiol Rev 87: 457–506, 2007.

373. Terentyev D, Cala SE, Houle TD, Viatchenko-Karpinski S,

Gyorke I, Terentyeva R, Williams SC, Gyorke S. Triadin over-expression stimulates excitation-contraction coupling and in-creases predisposition to cellular arrhythmia in cardiac myocytes.Circ Res 96: 651–658, 2005.

374. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Volpe P,

Williams SC, Gyorke S. Calsequestrin determines the functionalsize and stability of cardiac intracellular calcium stores: Mecha-nism for hereditary arrhythmia. Proc Natl Acad Sci USA 100:11759–11764, 2003.

375. Terentyev D, Viatchenko-Karpinski S, Valdivia HH, Escobar

AL, Gyorke S. Luminal Ca2� controls termination and refractorybehavior of Ca2�-induced Ca2� release in cardiac myocytes. Circ

Res 91: 414–420, 2002.376. Terentyev D, Viatchenko-Karpinski S, Vedamoorthyrao S,

Oduru S, Gyorke I, Williams SC, Gyorke S. Protein proteininteractions between triadin and calsequestrin are involved in mod-ulation of sarcoplasmic reticulum calcium release in cardiac myo-cytes. J Physiol 583: 71–80, 2007.

377. Tester DJ, Dura M, Carturan E, Reiken S, Wronska A, Marks

AR, Ackerman MJ. A mechanism for sudden infant death syn-drome (SIDS): stress-induced leak via ryanodine receptors. Heart

Rhythm 4: 733–739, 2007.378. Tocchetti CG, Wang W, Froehlich JP, Huke S, Aon MA, Wilson

GM, Di BG, O’Rourke B, Gao WD, Wink DA, Toscano JP,

Zaccolo M, Bers DM, Valdivia HH, Cheng H, Kass DA, Paolocci

N. Nitroxyl improves cellular heart function by directly enhancing

CALCIUM SPARKS 1543


on May 25, 2011


ownloaded from


cardiac sarcoplasmic reticulum Ca2� cycling. Circ Res 100: 96–104,2007.

379. Tour O, Adams SR, Kerr RA, Meijer RM, Sejnowski TJ, Tsien

RW, Tsien RY. Calcium Green FlAsH as a genetically targetedsmall-molecule calcium indicator. Nat Chem Biol 3: 423–431, 2007.

380. Tregear RT, Dawson AP, Irvine RF. Quantal release of Ca2�

from intracellular stores by InsP3: tests of the concept of control ofCa2� release by intraluminal Ca2�. Proc Biol Sci 243: 263–268, 1991.

381. Tsugorka A, Rios E, Blatter LA. Imaging elementary events ofcalcium release in skeletal muscle cells. Science 269: 1723–1726,1995.

382. Tu HP, Nelson O, Bezprozvanny A, Wang ZN, Lee SF, Hao YH,

Serneels L, De Strooper B, Yu G, Bezprozvanny I. Presenilinsform ER Ca2� leak channels, a function disrupted by familialAlzheimer’s disease-linked mutations. Cell 126: 981–993, 2006.

383. Valdivia HH, Kaplan JH, Ellis-Davies GC, Lederer WJ. Rapidadaptation of cardiac ryanodine receptors: modulation by Mg2�

and phosphorylation. Science 267: 1997–2000, 1995.384. Velez P, Gyorke S, Escobar AL, Vergara J, Fill M. Adaptation of

single cardiac ryanodine receptor channels. Biophys J 72: 691–697,1997.

385. Vest JA, Wehrens XH, Reiken SR, Lehnart SE, Dobrev D,

Chandra P, Danilo P, Ravens U, Rosen MR, Marks AR. Defec-tive cardiac ryanodine receptor regulation during atrial fibrillation.Circulation 111: 2025–2032, 2005.

386. Vinogradova TM, Maltsev VA, Bogdanov KY, Lyashkov AE,

Lakatta EG. Rhythmic Ca2� oscillations drive sinoatrial nodal cellpacemaker function to make the heart tick. Ann NY Acad Sci 1047:138–156, 2005.

387. Vinogradova TM, Zhou YY, Maltsev V, Lyashkov A, Stern M,

Lakatta EG. Rhythmic ryanodine receptor Ca2� releases duringdiastolic depolarization of sinoatrial pacemaker cells do not re-quire membrane depolarization. Circ Res 94: 802–809, 2004.

388. Volders PG, Kulcsar A, Vos MA, Sipido KR, Wellens HJ,

Lazzara R, Szabo B. Similarities between early and delayed after-depolarizations induced by isoproterenol in canine ventricularmyocytes. Cardiovasc Res 34: 348–359, 1997.

389. Wang SQ, Wei Cl Zhao GL, Brochet DXP, Shen JX, Song LS,

Wang W, Yang DM, Cheng HP. Imaging microdomain Ca2� inmuscle cells. Circ Res 94: 1011–1022, 2004.

390. Wang SQ, Song LS, Lakatta EG, Cheng H. Ca2� signallingbetween single L-type Ca2� channels and ryanodine receptors inheart cells. Nature 410: 592–596, 2001.

391. Wang SQ, Song LS, Xu L, Meissner G, Lakatta EG, Rios E,

Stern MD, Cheng H. Thermodynamically irreversible gating ofryanodine receptors in situ revealed by stereotyped duration ofrelease in Ca2� sparks. Biophys J 83: 242–251, 2002.

392. Wang SQ, Stern MD, Rios E, Cheng H. The quantal nature ofCa2� sparks and in situ operation of the ryanodine receptor arrayin cardiac cells. Proc Natl Acad Sci USA 101: 3979–3984, 2004.

393. Wang X, Weisleder N, Collet C, Zhou J, Chu Y, Hirata Y, Zhao

X, Pan Z, Brotto M, Cheng H, Ma J. Uncontrolled calcium sparksact as a dystrophic signal for mammalian skeletal muscle. Nat Cell

Biol 7: 525–530, 2005.394. Ward CW, Lederer WJ. Ghost sparks. Nat Cell Biol 7: 457–459,

2005.395. Ward CW, Protasi F, Castillo D, Wang Y, Chen SR, Pessah IN,

Allen PD, Schneider MF. Type 1 and type 3 ryanodine receptorsgenerate different Ca2� release event activity in both intact andpermeabilized myotubes. Biophys J 81: 3216–3230, 2001.

396. Ward CW, Schneider MF, Castillo D, Protasi F, Wang Y, Chen

SR, Allen PD. Expression of ryanodine receptor RyR3 producesCa2� sparks in dyspedic myotubes. J Physiol 525: 91–103, 2000.

397. Wegner F, Both M, Fink RH. Automated detection of elementarycalcium release events using the a trous wavelet transform. Bio-

phys J 90: 2151–2163, 2006.398. Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR,

Mohler PJ, Sun J, Guatimosim S, Song LS, Rosemblit N,

D’Armiento JM, Napolitano C, Memmi M, Priori SG, Lederer

WJ, Marks AR. FKBP12.6 deficiency and defective calcium releasechannel (ryanodine receptor) function linked to exercise-inducedsudden cardiac death. Cell 113: 829–840, 2003.

399. Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A,

Marks AR. Ryanodine receptor/calcium release channel PKAphosphorylation: a critical mediator of heart failure progression.Proc Natl Acad Sci USA 103: 511–518, 2006.

400. Weisleder N, Brotto M, Komazaki S, Pan Z, Zhao X, Nosek T,

Parness J, Takeshima H, Ma J. Muscle aging is associated withcompromised Ca2� spark signaling and segregated intracellularCa2� release. J Cell Biol 174: 639–645, 2006.

401. Weisleder N, Ferrante C, Hirata Y, Collet C, Chu Y, Cheng H,

Takeshima H, Ma J. Systemic ablation of RyR3 alters Ca2� sparksignaling in adult skeletal muscle. Cell Calcium 42: 548–555, 2007.

402. Weisleder N, Ma JJ. Ca2� sparks as a plastic signal for skeletalmuscle health, aging, dystrophy. Acta Pharmacol Sin 27: 791–798,2006.

403. Wellman GC, Nelson MT. Signaling between SR and plasma-lemma in smooth muscle: sparks and the activation of Ca2�-sensi-tive ion channels. Cell Calcium 34: 211–229, 2003.

404. Werner ME, Zvara P, Meredith AL, Aldrich RW, Nelson MT.

Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel. J Physiol 567: 545–556, 2005.

405. White C, McGeown JG. Carbachol triggers RyR-dependent Ca2�

release via activation of IP3 receptors in isolated rat gastric myo-cytes. J Physiol 542: 725–733, 2002.

406. Wickenden AD, Lee P, Sah R, Huang Q, Fishman GI, Backx

PH. Targeted expression of a dominant-negative K(v)4.2 K� chan-nel subunit in the mouse heart. Circ Res 85: 1067–1076, 1999.

407. Wier WG, Balke CW. Ca2� release mechanisms, Ca2� sparks,local control of excitation-contraction coupling in normal heartmuscle. Circ Res 85: 770–776, 1999.

408. Wier WG, Keurs HEDJ, Marban E, Gao WD, Balke CW. Ca2�

“sparks” and waves in intact ventricular muscle resolved by con-focal imaging. Circ Res 81: 462–469, 1997.

409. Wier WG. Calcium transients during excitation-contraction cou-pling in mammalian heart: aequorin signals of canine Purkinjefibers. Science 207: 1085–1087, 1980.

410. Wier WG, Cannell MB, Berlin JR, Marban E, Lederer WJ.

Cellular and subcellular heterogeneity of [Ca2�]i in single heartcells revealed by fura-2. Science 235: 325–328, 1987.

411. Wier WG, Egan TM, Lopez-Lopez JR, Balke CW. Local controlof excitation-contraction coupling in rat heart cells. J Physiol 474:463–471, 1994.

412. Wier WG, Kort AA, Stern MD, Lakatta EG, Marban E. Cellularcalcium fluctuations in mammalian heart: direct evidence fromnoise analysis of aequorin signals in Purkinje fibers. Proc Natl Acad

Sci USA 80: 7367–7371, 1983.413. Williams DA, Fogarty KE, Tsien RY, Fay FS. Calcium gradients

in single smooth muscle cells revealed by the digital imaging mi-croscope using Fura-2. Nature 318: 558–561, 1985.

414. Williams GS, Huertas MA, Sobie EA, Jafri MS, Smith GD. Aprobability density approach to modeling local control of calcium-induced calcium release in cardiac myocytes. Biophys J 92: 2311–2328, 2007.

415. Woo SH, Cleemann L, Morad M. Spatiotemporal characteristicsof junctional and nonjunctional focal Ca2� release in rat atrialmyocytes. Circ Res 92: 1–11, 2003.

416. Woo SH, Cleemann L, Morad M. Diversity of atrial local Ca2�

signalling: evidence from 2-D confocal imaging in Ca2�-buffered ratatrial myocytes. J Physiol 567: 905–921, 2005.

417. Woo SH, Risius T, Morad M. Modulation of local Ca2� releasesites by rapid fluid puffing in rat atrial myocytes. Cell Calcium 41:397–403, 2007.

418. Wu X, Bers DM. Sarcoplasmic reticulum and nuclear envelope areone highly interconnected Ca2� store throughout cardiac myocyte.Circ Res. In press.

419. Wu X, Zhang T, Bossuyt J, Li X, McKinsey TA, Dedman JR,

Olson EN, Chen J, Brown JH, Bers DM. Local InsP3-dependentperinuclear Ca2� signaling in cardiac myocyte excitation-transcrip-tion coupling. J Clin Invest 116: 675–682, 2006.

420. Xiao RP, Valdivia HH, Bogdanov K, Valdivia C, Lakatta EG,

Cheng H. The immunophilin FK506-binding protein modulatesCa2� release channel closure in rat heart. J Physiol 500: 343–354,1997.



on May 25, 2011


ownloaded from


421. Xiong W, Tian Y, Disilvestre D, Tomaselli GF. Transmuralheterogeneity of Na�-Ca2� exchange: evidence for differential ex-pression in normal and failing hearts. Circ Res 97: 207–209, 2005.

422. Xu L, Meissner G. Regulation of cardiac muscle Ca2� releasechannel by sarcoplasmic reticulum lumenal Ca2�. Biophys J 75:2302–2312, 1998.

423. Xu L, Tripathy A, Pasek DA, Meissner G. Potential for pharma-cology of ryanodine receptor/calcium release channels. Ann NY

Acad Sci 853: 130–148, 1998.424. Yamada J, Ohkusa T, Nao T, Ueyama T, Yano M, Kobayashi S,

Hamano K, Esato K, Matsuzaki M. Up-regulation of inositol 1,4,5trisphosphate receptor expression in atrial tissue in patients withchronic atrial fibrillation. J Am Coll Cardiol 37: 1111–1119, 2001.

425. Yan Y, Liu J, Wei C, Xie W, Wang Y, Cheng H. Bidirectionalregulation of Ca2� sparks by mitochondria-derived reactive oxygenspecies in cardiac myocytes. Cardiovasc Res 77: 432–441, 2008.

426. Yang HT, Tweedie D, Wang S, Guia A, Vinogradova T,

Bogdanov K, Allen PD, Stern MD, Lakatta EG, Boheler KR.

The ryanodine receptor modulates the spontaneous beating rate ofcardiomyocytes during development. Proc Natl Acad Sci USA 99:9225–9230, 2002.

427. Yao LJ, Wang G, Ou-Yang KF, Wei CL, Wang XH, Wang SR,

Yao W, Huang HP, Luo JH, Wu CH, Liu J, Zhou Z, Cheng HP.

Ca2� sparks and Ca2� glows in superior cervical ganglion neurons.Acta Pharmacol Sin 27: 848–852, 2006.

428. Yao Y, Parker I. Ca2� influx modulation of temporal and spatialpatterns of inositol trisphosphate-mediated Ca2� liberation inXenopus oocytes. J Physiol 476: 17–28, 1994.

429. Yaras N, Bilginoglu A, Vassort G, Turan B. Restoration ofdiabetes-induced abnormal local Ca2� release in cardiomyocytesby angiotensin II receptor blockade. Am J Physiol Heart Circ

Physiol 292: H912–H920, 2007.430. Yaras N, Ugur M, Ozdemir S, Gurdal H, Purali N, Lacampagne

A, Vassort G, Turan B. Effects of diabetes on ryanodine receptorCa release channel (RyR2) and Ca2� homeostasis in rat heart.Diabetes 54: 3082–3088, 2005.

431. Yasui K, Palade P, Gyorke S. Negative control mechanism withfeatures of adaptation controls Ca2� release in cardiac myocytes.Biophys J 67: 457–460, 1994.

432. Yin CC, Han HM, Wei RS, Lai FA. Two-dimensional crystalliza-tion of the ryanodine receptor Ca2� release channel on lipid mem-branes. J Struct Biol 149: 219–224, 2005.

433. Yin CC, Lai FA. Intrinsic lattice formation by the ryanodine re-ceptor calcium-release channel. Nat Cell Biol 2: 669–671, 2000.

434. Yu X, Chen XW, Zhou P, Yao LJ, Liu T, Zhang B, Li Y, Zheng H,

Zheng LH, Zhang CX, Bruce I, Ge JB, Wang SQ, Hu ZA, Yu HG,

Zhou Z. Calcium influx through I-f channels in rat ventricularmyocytes. Am J Physiol Cell Physiol 292: C1147–C1155, 2007.

435. Zahradnikova A, Zahradnik I, Gyorke I, Gyorke S. Rapid acti-vation of the cardiac ryanodine receptor by submillisecond calciumstimuli. J Gen Physiol 114: 787–798, 1999.

436. Zenisek D, Davila V, Wan L, Almers W. Imaging calcium entrysites and ribbon structures in two presynaptic cells. J Neurosci 23:2538–2548, 2003.

437. Zhang G, Fu Y, Yang D, Hao X, Tang Y, Lakatta EG, Wu C,

Cheng H. Contribution of spontaneous L-type Ca2� channel acti-vation to the genesis of Ca2� sparks in resting cardiac myocytes.Science in China 32–38, 2004.

438. Zhang X, Aman K, Hokfelt T. Secretory pathways of neuropep-tides in rat lumbar dorsal root ganglion neurons and effects ofperipheral axotomy. J Comp Neurol 352: 481–500, 1995.

439. Zhao GL, Zhao Y, Pan BX, Liu J, Huang XL, Zhang XQ, Cao

CM, Hou N, Wu CH, Zhao KS, Cheng HP. Hypersensitivity ofBKCa to Ca2� sparks underlies hyporeactivity of arterial smoothmuscle in shock. Circ Res 101: 493–502, 2007.

440. Zhou J, Brum G, Gonzalez A, Launikonis BS, Stern MD, Rios

E. Ca2� sparks and embers of mammalian muscle. Properties ofthe sources. J Gen Physiol 122: 95–114, 2003.

441. Zhou J, Brum G, Gonzalez A, Launikonis BS, Stern MD, Rios

E. Concerted vs sequential. Two activation patterns of vast arraysof intracellular Ca2� channels in muscle. J Gen Physiol 126: 301–309, 2005.

442. Zhou J, Launikonis BS, Rios E, Brum G. Regulation of Ca2�

sparks by Ca2� and Mg2� in mammalian and amphibian muscle. AnRyR isoform-specific role in excitation-contraction coupling? J Gen

Physiol 124: 409–428, 2004.443. Zhou J, Yi J, Royer L, Launikonis BS, Gonzalez A, Garcia J,

Rios E. A probable role of dihydropyridine receptors in repressionof Ca2� sparks demonstrated in cultured mammalian muscle. Am J

Physiol Cell Physiol 290: C539–C553, 2006.444. Zhou YY, Song LS, Lakatta EG, Xiao RP, Cheng H. Constitutive

beta2-adrenergic signalling enhances sarcoplasmic reticulum Ca2�

cycling to augment contraction in mouse heart. J Physiol 351–361,1999.

445. Zhou Z, Neher E. Calcium permeability of nicotinic acetylcholine-receptor channels in bovine adrenal chromaffin cells. Pflugers Arch

425: 511–517, 1993.446. ZhuGe R, Sims SM, Tuft RA, Fogarty KE, Walsh JV Jr. Ca2�

sparks activate K� and Cl� channels, resulting in spontaneoustransient currents in guinea-pig tracheal myocytes. J Physiol 513:711–718, 1998.

447. ZhuGe Rh DeCrescenzo V, Sorrentino V, Lai FA, Tuft RA,

Lifshitz LM, Lemos JR, Smith C, Fogarty KE, Walsh JV Jr.

Syntillas release Ca2� at a site different from the microdomainwhere exocytosis occurs in mouse chromaffin cells. Biophys J 90:2027–2037, 2006.

448. Zima AV, Blatter LA. Inositol-1,4,5-trisphosphate-dependent Ca2�

signalling in cat atrial excitation-contraction coupling and arrhyth-mias. J Physiol 555: 607–615, 2004.

449. Zou H, Lifshitz LM, Tuft RA, Fogarty KE, Singer JJ. ImagingCa2� entering the cytoplasm through a single opening of aplasma membrane cation channel. J Gen Physiol 114: 575–588,1999.

450. Zucker RS. The calcium-concentration clamp: spikes and revers-ible pulses using the photolabile chelator Dm-nitrophen. Cell Cal-

cium 14: 87–100, 1993.

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