modulation of the voltage sensor of l-type ca2+ channels by intracellular ca2+

The

Journ

al o

f G

ener

al P

hys

iolo

gy

555

J. Gen. Physiol.

© The Rockefeller University Press

•

0022-1295/2004/05/555/17 $8.00Volume 123 May 2004 555–571http://www.jgp.org/cgi/doi/10.1085/jgp.200308876

Modulation of the Voltage Sensor of L-type Ca

2

�

Channelsby Intracellular Ca

2

�

Dmytro Isaev, Karisa Solt, Oksana Gurtovaya, John P. Reeves,

and

Roman Shirokov

Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07101

abstract

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, ofL-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the con-centrations that are expected to be near an open channel (

�

100

�

M) completely and reversibly blocked calciumcurrent through L-type channels. Although charge movements associated with the opening (ON) motion of thechannel’s voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulseexperiments, the blockade of calcium current and the reduction of gating charge movements available for the sec-ond pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred onlyafter a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is lo-cated either in the channel’s central cavity behind the voltage-dependent gate, or it is formed de novo during de-polarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction ofthe OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously ob-served for voltage-dependent inactivation. Elevation of intracellular calcium concentration from

�

0.1 to 100–300

�

M sped up the conversion of the gating charge into the negatively distributed mode 10–100-fold. Since the “IQ-AA” mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium sim-ilarly to the wild-type, calcium/calmodulin binding to the “IQ” motif apparently is not involved in the observedchanges of voltage-dependent gating. Although calcium influx through the wild-type open channels does notcause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily ob-servable in the

�

1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels.We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediatesinactivation.

key words:

calcium channels • gating currents • calcium signaling

I N T R O D U C T I O N

Inactivation of L-type channels has traditionally beenattributed to two independent mechanisms initiated byeither intracellular calcium or membrane depolariza-tion (for reviews see Eckert and Chad, 1984; Hering etal., 2000; Budde et al., 2002). Local calcium concentra-tions on the intracellular side of a functioning calciumchannel rapidly change from 0.1

�

M at rest to

�

100

�

M when the channel is open. High intracellular cal-cium affects the gating of calcium channels, limitingtheir ability to reopen after a period of activity. A 25-yrsearch for the intracellular calcium-binding site respon-sible for inactivation (Brehm and Eckert, 1978) has cul-minated in the current view that calcium controls inac-

tivation by associating with a calmodulin molecule teth-ered to the channel (Lee et al., 1999; Peterson et al.,1999; Qin et al., 1999; Zuhlke et al., 1999). In L-typechannels, calcium binding to the carboxyl-terminallobe of the tethered calmodulin was proposed to ini-tiate inactivating transitions that may be linked to acomplex translocation of the calmodulin along the car-boxyl-terminal portion of the

�

1 subunit (Peterson etal., 1999). In cardiac L-type channels, the exclusive se-lectivity of calcium-dependent inactivation for the cal-cium ion (Lee et al., 1985) is a logical corollary of thecalmodulin-based theory.

When calcium channels pass ions other than cal-cium, or when they are blocked, they are thought to in-activate by a process that depends only on voltage.However, a current-dependent component of inactiva-tion of barium currents has also been suggested (Fer-reira et al., 1997). Decay of barium currents through in-activating calcium channels has multiexponential ki-netics, suggesting that voltage-dependent inactivationcomprises multiple, possibly interrelated (Hering et al.,2000) processes. Studies of channel inactivation basedon ionic current decay are complemented by analysis atthe level of gating currents.

The online version of this article contains supplemental material.

Dr. D. Isaev’s permanent address is Department of GeneralPhysiology, Nervous System, Bogomoletz Institute of Physiology,Ukrainian Academy of Science, 4 Bogomoletz St., 01024 Kyiv,Ukraine.

Address correspondence to Roman Shirokov, Department of Phar-macology and Physiology, New Jersey Medical School, UMDNJ, 185South Orange Avenue, Newark, NJ 07101-1709. Fax: (973) 972-7950;email: [email protected]

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Published April 26, 2004

http://jgp.rupress.org/content/suppl/2004/04/26/jgp.200308876.DC1.html Supplemental Material can be found at:

http://jgp.rupress.org/

http://jgp.rupress.org/content/suppl/2004/04/26/jgp.200308876.DC1.html

556

Inactivation of L-type Ca Channels

Gating currents, which are generated by chargedmoieties of voltage-gated channels, reflect directlythe transitions in closed and/or inactivated channels.When recorded in blocked channels, gating currentsare not distorted by the variable ionic environmentnear open channels. At the level of gating currents, in-activation of virtually all types of voltage-gated channelscan be described by two mechanisms with different ki-netics. A fast mechanism involves blocking by cytoplas-mic portions of the channel protein itself and charac-teristically “immobilizes” the gating charge (“ball-and-chain” mechanism) (Armstrong and Bezanilla, 1977;Hoshi et al., 1990; Bezanilla et al., 1991; Eaholtz et al.,1994). A slower (C-type) inactivation mechanism ap-pears to involve multiple conformational changes. Dur-ing slow inactivation, the intramembrane charge isnot immobilized but converts to a different modality(charge 2; Brum and Rios, 1987), which moves only atvoltages much more negative than those typical of thenoninactivated channels (charge 1). Charge 2 remainsmobile at negative voltage until the channel recoversvia slow transition(s) not limited by the charge move-ment itself. Gating currents in blocked cardiac L-typecalcium channels have been shown to inactivate by thecharge interconversion mechanism (Shirokov et al.,1992), because the return movement of the voltagesensors during recovery at very negative voltages occursmore rapidly than the recovery of ion currents. Fur-thermore, the onset of the reduction of charge mobileat positive voltages is slower than the inactivation decayof calcium, or barium, currents (Ferreira et al., 1997).Recent work of Ferreira et al. (2003) implied that in ad-dition to the charge 1/charge 2 interconversion, car-diac L-type channels also have a rapid component ofvoltage-dependent inactivation, which may be seen asimmobilization of the gating charge. It remains un-clear, however, how the two kinetic components of thechanges in gating currents during inactivation (immobi-lization vs

.

interconversion of charge) relate to the inac-tivation mechanisms of unblocked calcium channels.

Gating charge movements are not always well corre-lated with channel activity. For example, in virtually ev-ery experimental system, calcium currents throughL-type channels experience run-down during whole-cell patch-clamp recording. The process apparently in-volves multiple causes, including an influence of intra-cellular calcium (Belles et al., 1988; Costantin et al.,1999; Kepplinger et al., 2000; Rueckschloss and Isen-berg, 2001). At the same time, gating currents in L-typechannels are much more stable (Kostyuk et al., 1981;Hadley and Lederer, 1991b). In other words, voltagesensors remain mobile in the channels that do not passcalcium currents. As a second example, in native car-diac cells (Bean and Rios, 1989) and apparently in vari-ous expression systems, a substantial fraction of L-type

channels contribute to gating charge movement but donot open to pass calcium currents. This may reflect anegative regulatory control of channel opening by thedistal parts of the carboxyl-terminal segment of the

�

1

subunit (Gao et al., 2001), since certain carboxyl-termi-nal deletion mutants show normal ionic current ampli-tudes but much smaller gating charge movements thanwild-type channels (Wei et al., 1994). In the presentstudy, we use one such mutant (

�

1733) to investigatethe relation between calcium ions permeating thechannel and alterations in gating charge movements.

Previous attempts to demonstrate possible effects ofintracellular, or permeating, calcium ions on gating cur-rents in L-type calcium channels have yielded negativeor indirect results. Alterations in gating currents werenot detected during calcium flux through L-type cal-cium channels (Shirokov et al., 1993), or when flashphotolysis was employed to manipulate intracellular cal-cium (Hadley and Lederer, 1991a). More recent experi-ments, however, have revealed that gating currents maybe profoundly affected by calcium in the patch-clamp pi-pette solution (Ferreira et al., 1998), by calcium incom-ing through the channels (for N-type channels) (Shi-rokov, 1999), or by caffeine-induced calcium releasefrom the sarcoplasmic reticulum (Leroy et al., 2002).

In this report, we show that stationary elevation ofintracellular calcium to

�

100

�

M blocks L-type cal-cium channel activity and converts the voltage depen-dence of charge mobility to more negative values thanin noninactivated channels. Increasing concentrationsof calcium progressively accelerate the transition ofthe voltage sensor into these inactivated state(s). Usinga carboxyl terminus deletion mutant of the

�

1

subunit(

�

1733), we show that these inactivating transitionsare also induced by calcium permeating the openchannels; in this respect, the behavior of

�

1733 mu-tant is similar to that previously reported for N-typecalcium channels (Shirokov, 1999). Detailed analysisof the effects of intracellular calcium on the intramem-brane charge movements supports the view that the in-teraction site between calcium and the voltage sensoris isolated from the intracellular space in resting chan-nels and becomes accessible after a cooperative move-ment of several voltage sensors. We observed similar ef-fects of elevated cytosolic calcium in the “IQ-AA” muta-tion within the “IQ-motif”, where calmodulin bindingwas proposed to cause inactivation. This mutation hasbeen shown to eliminate the control of inactivation bycalmodulin (Zuhlke et al., 2000), tethered to the chan-nel molecule (Pitt et al., 2001). Taken together, our re-sults suggest that in addition to the calmodulin, cal-cium ions interact during inactivation with a site thatlies within the channel’s permeation pathway, or isformed after the opening movement of the voltagesensors.

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557

Isaev et al.

M A T E R I A L S A N D M E T H O D S

The following proteins were transiently expressed in tsA-201cells: CaV1.2 channel, as a combination of

�

1C

(Entrez pro-tein database accession no. CAA33546),

�

2a

(AAK14821),

�

2a

(AAA81562) subunits, and the NCX1.1 (AAA30509) sodium–cal-cium exchanger (Aceto et al., 1992). The cDNAs were inpcDNA3.1 mammalian expression vector (Invitrogen). Muta-tions were done by PCR overlap extension, or by the Quick-Change site-directed mutagenesis kit (Stratagene) and verifiedby sequencing. In the “IQ-AA” mutant, I1654A and Q1655Aamino acid mutations were combined with amino-terminal dele-tion

�

134 and carboxyl-terminal deletion

�

1733.Cells were transfected by a modified calcium-phosphate–based

transfection procedure using 20

�

g of each DNA per 60-mm cul-ture dish, as described previously (Shirokov et al., 1998). Selec-tion of the expressing cells was aided by EYFP fluorescence (1–3

�

g per dish, pEYFP-N1 vector; CLONTECH Laboratories, Inc.).Expression of NCX was assessed by the presence of a characteris-tic outward steady-state current (at least 2 pA/pF at the holdingpotential of

90 mV) observed when NCX worked in the re-versed mode (Fig. S1). The exchanger current had little effecton the measurement of asymmetric ionic and gating currents(Fig. S2). Online supplemental figures available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1.

Composition of solutions is given at Table I. We used chlorideas the main anion as it has minimal Ca

2

�

buffering capacity. FreeCa

2

�

in the pipette solution was adjusted by adding CaCl

2

andmeasured by a Ca

2

�

-selective electrode (WPI, Inc.). All solutionswere adjusted to 320 mosmol·kg

1

and pH 7.3. Experiments wereperformed at room temperature. Except for the experiment il-lustrated in Fig. 1, recordings started 5–8 min after establishingthe whole-cell configuration. The delay was sufficient for calciumcurrents to stabilize after the initial run-up.

Symmetric capacitive and leak currents were subtracted elec-tronically by capacitance compensation circuitry of the patch-clamp amplifier (Axopatch 200B; Axon Instruments, Inc.). Wechose small cells and used relatively large pipettes (0.8–1.2 M

�

).Cell capacitance (11

�

5 pF,

n

126), series resistance (R

s

6.4

�

3.1 M

�

) and leak resistance were set during a controlpulse applied from the holding potential of

90 to

100 mV. Noadditional subtraction of symmetric currents were required to

record gating currents. Previously (Shirokov et al., 1998), wedemonstrated the validity of this approach, which works well forthe small cells we used.

To calculate charge transfer, the current transient was inte-grated after subtraction of the steady-state component. Thesteady-state component was determined as a 10 ms average, 50ms after the beginning of the voltage pulse. Curve fitting wasdone by a nonlinear least-squares routine of SigmaPlot (SPSS,Inc.). Data are presented as means

�

SEM.

Online Supplemental Material

The additional material (available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1) describes the following: tech-nical aspects of the experiments in cells expressing NCX and Cachannels (Figs. S1 and S2), estimation of the magnitude of gat-ing charge during voltage pulses to the reversal potential (Fig.S3), comparison of activation/deactivation kinetics of whole-cellCa

2

�

currents in the

�

1733 mutant and in the wild-type channel(Figs. S4 and S5), and a consideration of a three-particle alloste-ric model explaining why Ca

2

�

binding to a low-affinity site mightpreferentially affect the onset, but not the recovery, kinetics(Fig. S6).

R E S U L T S

High Cytosolic Calcium Blocks Ionic Current and Affects Channel Gating

We examined the effects of directly perfusing high cal-cium concentrations through the whole-cell patch pi-pette on ionic and gating currents in L-type calciumchannels. As illustrated in Fig. 1, when the pipette con-tained 0.3 mM calcium, calcium currents initially in-creased during the first several minutes of perfusion(trace

2

), but were completely blocked by 18 min (trace

3

). After ionic currents became very small, gating cur-rents could be recorded directly, i.e., without any ioniccurrent blocker. The outward ON transients of the in-tramembrane charge movement appeared to be unaf-fected by the calcium load. However, the OFF tran-sients were dramatically reduced. The asymmetric ef-fect of calcium on gating currents suggests that calciuminteracts with the channel only after the opening move-ments of the voltage sensors (see below).

The reduction of the OFF transient could be clearlyobserved only when most of the channels were blockedby intracellular calcium so that ionic tail currents weresmall. Thus, it was impossible to correlate directly thereduction of calcium currents with the reduction in theOFF gating current transient throughout the range ofresults. To overcome this difficulty, we designed a two-pulse experiment, in which we compared the effects ofcalcium load on the magnitude of ionic currents dur-ing the first pulse and on the amount of intracellularcharge movement of charge 1 type available for the sec-ond pulse. The reduction in OFF currents would bemanifest as a loss of charge 1 availability, which in turnwould be reflected in a reduction in the ON gating cur-rent transient during the second pulse.

T A B L E I

Composition of Solutions

Chemical

Name Ca

2

�

NaCl CsCl TEA-Cl Tris-Cl HEPES EGTA Mg-ATP GdCl

3

mM mM mM mM mM mM mM mM mM

Pipette

1 EGTA

— — 155 — — 10 1 5 —

0.01 Ca

0.01

a

— 155 — — 10 — 5 —

0.1 Ca

0.1

a

— 155 — — 10 — 5 —

0.3 Ca

0.3

a

— 155 — — 10 — 5 —

0 Ca, 20 Na

— 20 130 — — 10 10 5 —

Bath

0 Na

10

b

— — 150 10 — — — —

150 Na

10

b

150 — — 10 — — — —

150 Na, Gd

10

b

150 — — 10 — — — 0.015

Gd

10

b

— — 150 10 — — — 0.015

a

CaCl

2

was added for the indicated concentration of free Ca

2

�

, as mea-sured by Ca-selective electrode.

b

CaCl

2

was added for the indicated total concentration of Ca

2

�

.

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558

Inactivation of L-type Ca Channels

The pulse protocol is illustrated in Fig. 2 A (top).The first pulse to

�

20 mV activated nearly maximal cal-cium currents. Then, the voltage went to

50 mV for20 ms. Although the tail currents decayed to zero bythe end of the interpulse, indicating that the channelshad closed, the interpulse was too brief to allow recov-ery from the inactivation elicited by the first depolariza-

Figure 1. Ca2� load through thepatch pipette blocked Ca2� currentthrough L-type Ca2� channels and re-duced their OFF gating current. Cur-rent traces were elicited by voltagepulses from the holding potential from90 to 20 mV. Pipette solution had 0.3mM of free Ca2� (0.3 Ca, Table I), andbath solution was 0 Na.

F

igure

2. Comparison of Ca

2

�

current magnitude and availabil-ity of the intramembrane charge movement measured in a double-pulse experiment. (A) Currents elicited by the pulse protocolshown at the top were recorded in a cell loaded with Ca

2

�

throughthe patch pipette. Pipette solution had 0.1 mM of free Ca

2

�

(

0.1Ca

), and bath solution was

0 Na

. The time after establishing thewhole-cell recording configuration is indicated near traces. Ca

2

�

current at the first pulse to

�

20 mV and gating currents elicited bythe second pulse to the reversal potential of ionic currents weredecreasing in parallel during the course of experiment. (B) Cur-rents obtained similarly to those shown in A, but in a cell recordedwith

1 EGTA

pipette solution. While Ca

2

�

currents at the first pulseto

�

20 mV slowly run-down, gating currents at the second pulse tothe reversal potential did not change. (C) Correlation between themagnitude of Ca

2

�

current and the gating charge during the sec-ond pulse for the cell shown in A. The gating charge was obtainedby integrating the ON transient during the second pulse, as de-scribed in the text. Linear regression is shown by the thin line, thedashed lines show 99% prediction interval. (D) Correlation be-tween the magnitude of Ca

2

�

current and the gating charge dur-ing the second pulse for the cell shown in B. Lines are as in C.

tion. To minimize ionic currents, the voltage appliedduring the second pulse was very close to the reversalpotential for the particular cell under study. The rever-sal potential varied from 50 to 70 mV in different cells,and it generally drifted by 3–5 mV during the time ofexperiment. Once the voltage of the second pulse waschosen, it remained the same during the experiment.For the cells illustrated in Fig. 2, A and B, the secondpulse went to 53 and 68 mV, respectively. Since ioniccurrents were relatively small and slow during the sec-ond pulse, the ON gating current transient at the sec-ond pulse was clearly resolvable. When the cell was re-corded with

0.1 Ca

pipette solution (Fig. 2 A), calciumcurrents during the first pulse and the ON gating cur-rents during the second pulse each became progres-sively smaller with the time of recording. As expected,it took much longer for calcium currents to run downwhen the pipette contained

1 EGTA

solution (Fig. 2 B).Importantly, however, the gating current transients atthe second pulse were much more stable and did notdecay despite the eventual loss of ionic current. The re-sults provide qualitative evidence that calcium loadingexerts similar effects on ionic and gating currents.

A quantitative analysis of these results requires ameans of quantifying the ON charge movement underthe conditions of these experiments. In the absence ofionic current blocker, however, it is impossible to elimi-nate the contribution of residual ionic currents to thecharge movement during the second pulse. Therefore,we estimated the bulk value of the gating charge by in-tegrating the ON gating current transient after sub-tracting the 2-ms current average near the minimalvalue reached 4–5 ms after the beginning of the second

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559

Isaev et al.

pulse. Most of the gating charge had moved at this timeand the slowly developing outward current did not sig-nificantly obstruct the transient. This procedure is illus-trated in Fig. S3, available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1.

Fig. 2, C and D, displays the correlations betweenionic currents and the estimated ON charge movementduring the period in which the cells were perfused withor without 0.1 mM calcium. As shown in Fig. 2 C for thecell loaded with 0.1 mM calcium, the gating charge atthe second pulse (from

50 mV to the reversal poten-tial) went almost to zero in parallel with the reductionof calcium current at the first pulse. In four cells testedwith

0.1 Ca

pipette solution, the linear regression,Q(V

rev

)

A·I

peak

(20 mV)

�

B, had the

r

2

value of atleast 0.89, the slope A was

0.47

� 0.07 fC/pA, and theintercept B was 25 � 62 fC, a value not significantly dif-ferent than zero. Fig. 2 D illustrates comparable resultsfor the cells with 1 EGTA pipette solution. In this case,there was practically no loss in gating charge even afterionic currents had run down to near zero. For the fivecells perfused with 1 EGTA, the linear regressions werealso of a good quality (the r2 value was at least 0.92), butthe slope was only 0.07 � 0.03 fC/pA, and the originof coordinates was never within the 99% interval forprediction. These results agree with previous findingsthat calcium currents recorded in cells with EGTA inthe pipette run down to zero without a significantchange in gating currents (Kostyuk et al., 1981; Hadley

and Lederer, 1991b). Overall, the results demonstratethat during calcium loading, the calcium current dur-ing the first pulse and the gating charge available to thesecond pulse were reduced in parallel (Fig. 2 C). We in-fer that calcium affected both ionic and gating currentsby a common mechanism.

Alteration of Cytosolic Calcium by Sodium–Calcium Exchange

Inhibition of channel activity during direct perfusion withcalcium required long perfusion times (13 � 6 min with0.3 Ca pipette solution, n 14). This probably reflectsthe influence of calcium buffering and organellar seques-tration on calcium diffusion into the cell, since 90% equil-ibration of calcium between the pipette and a 20-pF cellwould occur within only 2 min if diffusion of calciumwere free (Oliva et al., 1988; Pusch and Neher, 1988). Thelong perfusion times could also be compatible with elabo-ration of a second messenger, or the activation of pro-teases or other degradative enzymes. Thus, it would be de-sirable to alter the calcium concentration in the vicinity ofthe channel by a process that was both rapid and readilyreversible. For this purpose, we transfected cells with thecardiac sodium–calcium exchanger NCX1.1 (Aceto etal., 1992), so that submembrane calcium concentrationscould be rapidly and reversibly altered by changing thesodium gradient across the membrane.

The effects of exchange activity on channel functionare illustrated in the experiment in Fig. 3 A. Here, cal-

Figure 3. Effects of manipulations ofintracellular Ca2� by sodium–calciumexchanger (NCX) on L-type Ca2� chan-nels. Voltage pulses were from 90 to20 mV. (A) Activation of NCX reversesthe inhibition of Ca2� current by intra-cellular Ca2�. Pipette solution was 0.3Ca. When bathing solution was changedfrom 0 Na (indicated by open boxes) to150 Na (indicated by gray boxes), NCXreduced local [Ca2�]. (B) NCX func-tioning in the reverse mode reversiblyblocks Ca2� currents. Pipette solution: 0Ca, 20 Na. When bathing solution waschanged from 150 Na to 0 Na, NCX in-creased local [Ca2�].

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560 Inactivation of L-type Ca Channels

cium (0.3 mM) was loaded through the pipette intocells expressing both L-type channels and NCX. At first,neither the pipette nor the bathing solution containedsodium and under these conditions the exchanger wasnot active. After 3 min of loading, calcium currentswere partially inhibited (trace 1). When the bathing so-lution was changed to that with 150 mM sodium, cal-cium current rapidly increased (trace 2). In four simi-lar experiments, current increased by an average of112 � 16% within 20 s after the switch to 150 Na. Whenexternal sodium was removed for a longer period oftime, ionic and gating currents became similar to thoseobserved previously in calcium-loaded cells (comparetrace 3 in Fig. 1 and Fig. 3 A). Finally, when NCX wasagain activated by 150 mM sodium, the ionic currents(trace 4) were partially restored. Extracellular sodiumhad little or no effect on channel currents in controlexperiments in cells without NCX (4 � 3%; n 5).Therefore, the sodium-dependent increase in calciumcurrents reflected the efflux of intracellular calcium bysodium–calcium exchange. The rapidity of the restora-tion of calcium currents, in the face of continued cellu-lar perfusion with 0.3 mM calcium, suggests that theexchanger’s effects are local, i.e., exchange activity rap-idly alters the calcium concentration in a submem-brane space sensed by the calcium channel.

NCX was also used to load calcium into the cell (Fig.3 B). Here, the pipette solution had 20 mM sodium

and 10 mM EGTA and the bath solution had 150 mMsodium. Under these conditions, calcium currents werelarge (trace 1). Removing sodium from the bath solu-tion induced a biphasic decline in channel currents ascalcium entered the cell in exchange for intracellularsodium. In this experiment, the calcium currents ini-tially declined by about one-half within the time be-tween two consecutive pulses (20 s, trace 2) and thendecreased more slowly to nearly zero during the ensu-ing 5 min (trace 3). At this time, the OFF gating cur-rent was greatly reduced (trace 3), indicating that theexchanger had increased intracellular calcium suffi-ciently to reproduce the effects seen during direct per-fusion with high calcium pipette solutions. Readditionof sodium to the bath caused a substantial increase ofcalcium current (trace 4). Removing extracellular so-dium a second time again made calcium currents smalland the gating currents asymmetric (trace 5). Simi-lar results were obtained in seven other cells selectedfor measurable NCX currents (see materials andmethods).

The mechanisms underlying the biphasic timecourse of calcium current decline are unclear. One pos-sibility is the presence of two calcium-dependent block-ing mechanisms with different calcium affinities. Alter-natively, diffusion of calcium away from the membraneand the interaction with cytosolic EGTA may accountfor the peculiar time course. Yet another possibility is

Figure 4. Intracellular Ca2� loadedthrough NCX reversibly blocked Ca2�

currents and reduced the OFF gatingcurrent transients. Voltage pulses werefrom 90 to 20 mV. Pipette solutionwas 0 Ca, 20 Na. (A) Protocol of applica-tion of bath solutions (boxes) and am-plitudes of Ca2� currents (dots). Whenbathing solution was changed from 150Na (gray boxes) to 0 Na (open boxes),NCX functioning in the reversed modeincreased local [Ca2�]. When bathingsolution was 150 Na, Gd, Ca2� currentswere blocked. (B) Current traces re-corded at times indicated by corre-sponding numbers in A.

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561 Isaev et al.

that calcium influx through NCX decreased (“inacti-vated”) with time. Regardless of the precise reasons forthe kinetics of this response, one point is clear: bothionic currents and the OFF transients were affected bycalcium influx through NCX in the same manner as bycalcium load through the pipette. The reversible andmore rapid action of calcium delivered by NCX, ratherthan by the pipette, speaks for a direct, diffusion-lim-ited mechanism.

The reversibility of calcium’s effects on gating cur-rents is documented in the experiment shown in Fig. 4.Calcium was loaded into the cell by the reverse mode ofthe NCX under conditions where the pipette had 20mM sodium and 10 mM EGTA. When sodium in thebath solution was replaced with TEA, calcium currentswere reduced to zero and gating currents became asym-metric (compare traces 1 and 2). When sodium was re-stored to the bath medium, calcium currents partiallyrecovered (trace 3). At this point, we blocked the re-stored calcium currents with a brief application of 15�M gadolinium (trace 4). The remaining gating cur-rent transients were symmetric, as expected when gat-ing currents are recorded at low intracellular calcium(e.g., with 1 EGTA pipette solution). After partial recov-ery in gadolinium-free solution (trace 5), extracellularsodium was again replaced with TEA. The resulting in-flux of calcium through the exchanger again greatlyreduced calcium currents through calcium channels(trace 6). Blockade of these currents with gadoliniumrevealed an asymmetric reduction in the OFF gatingcurrent transient (trace 7). Addition of sodium to thebath again restored both calcium currents (traces 8and 10) and the symmetry of the gating current tran-sients (trace 9).

The effects of calcium loading through reverse ex-change activity are summarized as follows. Calcium cur-rents were reduced to 10% of their original valuewithin 6 � 1 min, n 12, of calcium load throughNCX. This was significantly faster than current run-down in the absence of intracellular calcium: With 0 Ca20 Na solution in the pipette and 150 Na solution in thebath, it took 39 � 12 min, n 18, for calcium currentsto run down to 50% of their maximal magnitude, if in-tracellular calcium was not manipulated. In five cellstested for the effect of calcium load through NCX ongating currents, determined in the presence of gadolin-ium as illustrated in Fig. 4, elevation of intracellular cal-cium reduced the ratio of the OFF charge to the ONcharge (QOFF/QON) to 0.21 � 0.06. Readdition of so-dium to the bath restored only 48 � 9% of calcium cur-rent magnitude before the second removal of extracel-lular sodium (compare traces 1 and 4 in Fig. 3 A, ortraces 1 and 3 in Fig. 4). However, QOFF/QON was equalto 0.97 � 0.06, n 5, when the restored calcium cur-rents were blocked by gadolinium. Given that elevation

of intracellular calcium by perfusion through the pi-pette, or by NCX, rapidly and reversibly affected bothionic and OFF gating currents, it is likely that both theavailability of ionic currents and the voltage-dependentgating are controlled by the same calcium-dependentmechanism.

Cytosolic Calcium Shifts the Voltage Dependence of QOFF

An analysis of the voltage dependence of calcium’s ef-fects on intramembrane charge movement providedfurther insights into the mechanism and location of theinteraction between calcium and voltage sensors. Forthe experiments illustrated in Figs. 5–7, we blockedionic currents with 15 �M gadolinium and studied thechanges in voltage distribution of the intramembranecharge movement that accompany combined applica-tion of high intracellular calcium and depolarization.Fig. 5 A shows representative traces for experimentsconducted with either 1 EGTA or 0.1 Ca solutions in theperfusion pipette, and Fig. 5 B compares voltage de-pendencies for the ON charge (circles) in pipette solu-tions with different calcium concentrations. Before av-eraging, the individual ON charge transfer functionswere normalized in each cell by the maximal charge(Qmax), which was determined by fitting the Boltzmanndistribution Q Qmax/(1 � exp((V V1/2)/K)) tothe values of the ON charge. The solid lines in panel Bare the best fits to the averaged values. Loading of 0.3mM calcium negatively shifted the voltage distributionof the ON charge movement by �20 mV. This wasprobably due to the screening of the surface charge.The symbols in panel C represent the averaged OFFcharges under several different perfusion conditions.Before averaging, the OFF charge values were normal-ized in each cell by the Qmax, which was determinedfrom the ON transients as described above. The OFFtransients recorded during repolarizations to 90 mVwere dramatically reduced in high calcium if the testpulse went more positive than 10 mV. The dashedlines in panel C are the fits to the ON charge transferfunctions in panel B. In calcium-loaded cells, they devi-ated significantly from the OFF charge transfer func-tions at potentials above 0 mV. The difference betweenthe ON and the OFF charges is plotted in panel D. Theeffect of calcium, as judged by the apparent loss of themobile charge in the OFF transient, had a more posi-tively distributed dependence on voltage when com-pared with that of the ON charge and a significantlysteeper slope (K 11 vs. 25 mV).

When the pipette solution contained 0.3 mM cal-cium, the OFF transients could be recovered if the cellwas repolarized to very negative voltages. This is illus-trated in Fig. 6 A, where the OFF transient carriedabout half of the charge in the ON transient when themembrane was repolarized to 120 mV. During repo-

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larization to 200 mV, almost all the OFF charge couldbe recovered (Fig. 6 A, bottom trace). The voltage dis-tributions of the OFF charge (QOFF) were derived as fol-lows. The QOFF charge transfer function in each cell wasfit by: Q Q∞ � Qmax /(1 � exp((V V1/2)/K)).Then, the (QOFF Q∞)/Qmax values were averaged.As shown in Fig. 6 B, the voltage distribution of theOFF charge movement recorded after a 20-ms depo-larization to 20 mV was shifted by about 90 mV. Thereduction of the OFF charge measured in the experi-ments shown in Fig. 5 is a manifestation of the sameeffect. A similar negative shift in the voltage depen-dence of charge movement was observed with variousvoltage-gated channels in the absence of calcium, i.e.,during voltage-dependent inactivation (Bezanilla etal., 1982; Brum and Rios, 1987; Shirokov et al., 1992,1998; Olcese et al., 1997). However, in the absenceof calcium, the shift in charge movement requiredmuch longer depolarizations. For example, in car-diac calcium channels recorded with a low-calcium in-tracellular solution, the full conversion of the chargemovement into the negatively distributed mode re-quired several seconds of depolarization to voltagesabove 0 mV (Shirokov et al., 1998). In contrast, inour experiments with high intracellular calcium, two-thirds of the charge moved more negatively than 80mV after only 20 ms of depolarization to 20 mV (Fig.6 B).

Additional information on time dependence of thecalcium’s effects on charge movement is presented inFig. 7, where we measured reduction in OFF chargemovement after depolarizations of different duration.Because the return voltage was 60 mV, most of the re-corded charge was of the positively distributed mode.The OFF transients shown in Fig. 7 A are spaced ac-cording to the duration of the preceding conditioningat 20 mV. The transients recorded after a depolariza-tion �5 ms were progressively smaller for increasingconditioning duration. With the 1 EGTA pipette solu-tion, the reduction was slow and had the time constantof 0.7 s, somewhat less than reported previously for 10mM EGTA (2.2 s; Shirokov et al., 1998). The reductionof the mobile charge was �10 times faster when cellswhere loaded with 0.1 mM calcium (77 ms) and �100times faster (8 ms) with 0.3 mM calcium in the pipette.The more rapid reduction of the OFF transients at 60mV in high intracellular calcium was not accompaniedby a detectable slowing of their kinetics. Also, therewas no slow component significantly deviating fromthe baseline over the 50 ms recording interval at 60mV. Since charge immobilization, or rapid inactivation(Ferreira et al., 2003), would be characterized by ameasurable slow component of charge movement co-temporal with recovery from inactivation, we concludethat this indicates that there was no detectable immobi-lization of charge by calcium under our experimental

Figure 5. OFF gating currents elic-ited by repolarizations to 90 mV werereduced by intracellular Ca2�. Bathingsolution: Gd. Pipette solutions are indi-cated in C. (A) Representative tracingsfor pulses to 40, 0, 40, and 80 mV. (B)Average ON charge transfer functionsobtained with different pipette solu-tions, as illustrated in A. The averagingprocedure is described in the text. Dif-ferent symbol sizes correspond to differ-ent [Ca2�], as listed in C. Only the max-imal SEM value is shown for clarity. Thecurves are single Boltzmann fits. V1/2

was: 15 � 3 mV (1 EGTA), 13 � 4 mV(0.01 Ca), 7 � 3 mV (0.1 Ca), and 5 �5 mV (0.3 Ca); K was 25 � 3 mV. Eachgroup had n 6 cells. (C) Average OFFcharge transfer functions. The dashedlines are the fits to the ON charge trans-fer from B. (D) The difference betweenON and OFF charge transfer functions.The solid lines are single Boltzmannfits. V1/2 was: 31 � 12 mV, and K was11 � 3 mV for all fits.

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conditions. Additional support to this view comes fromthe analysis of the kinetics of the OFF transients at verynegative voltages (like those shown in Fig. 6 for 200mV). In contrast to control traces, the decay of the OFFtransients at 200 mV in cells recorded with 0.1 mMcalcium in the pipette was biexponential with a signifi-cant slow component (� 7.4 � 1.1 ms, n 4). Thistime constant is similar to that of the charge 2 move-ment in cardiac cells (Shirokov et al., 1992). Recoveryof calcium currents takes much longer at 200 mV(� 46 � 8 ms, n 4, after 100 ms conditioning to 20mV; unpublished data). Thus, the bulk of the in-tramembrane charge (Fig. 6) moves at 200 mV be-fore the channels become able to open. Taken to-gether, the data in Figs. 5–7 indicate that intracellular

calcium acts on the channel by accelerating the sametransitions that occur during voltage-dependent inacti-vation (charge 1/charge 2 interconversion).

The Effects of Calcium Are Independent of Calmodulin

To test whether calmodulin was involved in the observa-tions described above, we used the “IQ-AA” mutant. Inthis mutant, the key amino acids I1654 and Q1655 ofthe so-called “IQ” motif, where calcium/calmodulin isthought to bind and cause inactivation, were mutatedto alanines. As expected from the work of Zuhlke et al.(2000), calcium currents through the mutant channelinactivated slowly, with kinetics similar to those of bar-ium currents. As shown in Fig. 8, when intracellular cal-cium was increased by reverse exchange activity in cells

Figure 6. Combination of high intra-cellular [Ca2�] and a brief depolariza-tion caused a large negative shift of thevoltage distribution of the intramem-brane charge movement in L-type Ca2�

channels. (A) Representative tracingsof the OFF charge for repolarizations to40, 40, 120, and 200 mV appliedafter a 20-ms prepulse to 20 mV. Bath-ing solution: Gd. Pipette solutions areindicated. (B) Average OFF charge dis-tributions obtained with 1 EGTA (opencircles), or 0.3 Ca (filled circles) pipettesolutions, as illustrated in A. The curvesare single-Boltzmann fits. In 1 EGTA so-lution: V1/2 27 � 2 mV, K 27 � 3mV, n 6. With 0.3 Ca solution: V1/2 116 � 6 mV, K 29 � 5 mV, n 4.

Figure 7. Intracellular Ca2� acceler-ates inactivation of gating currents.Bathing solution: Gd. Pipette solutionsare indicated. (A) Representative OFFgating current traces elicited by repolar-izations to 60 mV are spaced accord-ing to the time of conditioning at 20mV. (B) Average charge transferredduring the OFF transients plotted ver-sus logarithm of conditioning duration.Different symbols correspond to differ-ent [Ca2�] in the pipette. Charge trans-fers are normalized to the maximalvalue in each cell and averaged, n 4for 0.3 Ca, n 7 for other concen-trations. The maximal values wereachieved at 10 ms with 1 EGTA and 0.01Ca, at 5 ms with 0.1 Ca, and at 2.5 mswith 0.3 Ca pipette solutions. Only themaximal SEM value is shown for clarity.Curves are single-exponential fits. Thetime constants were: 670 � 47 ms in 1EGTA, 280 � 35 ms in 0.01 Ca, 77 � 12ms in 0.1 Ca, and 8 � 2 ms in 0.3 Ca.

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expressing the “IQ-AA” mutant (n 5), both ionic andthe OFF gating currents were reduced in the samemanner as in the wild-type channel (Figs. 3 and 4). Cal-cium loading by direct perfusion also blocked ionicand the OFF gating currents in the “IQ-AA” mutant(unpublished data; n 4). Thus, calcium/calmodulinbinding to the “IQ” motif does not promote thechanges in voltage-dependent gating seen at highcalcium.

Calcium Permeating the Channel Affects Gating Currents in the �1733 Mutant

It is possible that the effects of intracellular calciumload on the intramembrane charge movement are ex-erted only when calcium increases are long lasting rela-tive to the channel-gating kinetics and that the calciumions actually passing through the channel may not actthe same way. Our previous attempts to demonstratethat calcium currents through L-type channels affectedtheir gating currents were unsuccessful (Shirokov et al.,1993). While the negative observations were in accordwith the widely held view that calcium- and voltage-de-pendent components of inactivation have distinct mo-lecular mechanisms, we also proposed a trivial explana-tion. Both native cardiac cells and expression systemsusually produce a large number of calcium channelmolecules that contribute to the gating current but donot open to pass calcium into the cell (Bean and Rios,1989). Because gating currents are recorded from allchannels, while only a fraction of channels open, thelocal effect of incoming calcium could go undetected.

The dissociation between ionic conductance andvoltage-dependent gating in calcium channels was orig-inally thought to reflect the properties of the mecha-nism by which calcium channels run down (Kostyuk etal., 1981; Hadley and Lederer, 1991b). However, in viewof more recent studies of the negative control of L-typechannels by the distal parts of the carboxyl terminus oftheir pore-forming �1 subunit (Wei et al., 1994; Gao et

al., 2001), we propose that the excess of the voltage sen-sors is not due to ionic current run-down, but isbrought about by the interactions involving the car-boxyl terminus. Carboxyl terminus deletions, which donot remove the sequence above position 1700 andleave calmodulin binding domains untouched, pro-duce channels that pass calcium currents of similar orgreater magnitude than the wild-type, but their gatingcurrents are much smaller. Based on the relationship:I N · PO · is.sh., where I is the whole-cell current, N isthe number of channels available to be open, PO is theprobability of a channel being open, and is.sh. is the cur-rent through an open channel, Wei et al.(1994) pro-posed that carboxyl terminus deletion mutants havehigher PO, since is.sh. was not changed and the maximalgating charge (hence, N) was less. However, an in-crease in PO must be accompanied by changes in the ki-netics of channel opening and/or closing. Both thepublished works of others (Wei et al., 1994; Gao et al.,2001) and our experiments with the carboxyl terminusdeletion mutant �1733 (see Figs. S4 and S5) demon-strate that the activation/deactivation kinetics of themacroscopic calcium currents through the carboxylterminus deletion mutants are virtually indistinguish-able from those of the wild-type. Since the opening/closing kinetics (hence, PO) are not altered signifi-cantly in the deletion mutant, the mutation increasesthe number of channels available for opening, ratherthan PO. This also means that in the mutant, unlike inthe wild-type, gating currents are generated mostly bythe channels that pass calcium.

Encouraged by our previous finding that calciumcurrent through N-type channels does in fact acceler-ate the appearance of the negative shift in the voltagedependence of the intramembrane charge movements(Shirokov, 1999), we did similar experiments on the�1733 channels, which, as discussed above, have higheravailability for opening. The pulse protocol shown onthe top of Fig. 9 A allowed us to monitor ionic currentsand the intramembrane charge movement in inacti-vated channels. First, the conditioning pulse activatedcalcium current. Then, the membrane was briefly repo-larized to 150 mV, and a test pulse to 20 or 50 mVwas applied. The magnitude of calcium current duringthe test pulse to 20 mV (trace 1) reflected the numberof noninactivated channels at the moment when thetest pulse was applied. If the test pulse went to 50 mV(trace 2), it moved little charge in noninactivated chan-nels, because most of the charge in noninactivatedchannels is mobile at voltages more positive than 50mV (Fig. 5). However, because inactivation is accompa-nied by a negative shift in the voltage distribution ofthe intramembrane charge movement (see Fig. 6; alsoShirokov et al., 1998), an increase of the charge move-ment elicited by the pulse from 150 to 50 mV would

Figure 8. The “IQ-AA” mutant, in which Ca2�/calmodulin doesnot accelerate inactivation of Ca2� current, was affected in thesame manner as the wild-type channel. Both ionic and OFF gatingcurrents were reduced by the Ca2� load. Experimental protocolwas as in Fig. 3 B.

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provide evidence that inactivation during the condi-tioning pulse had affected the voltage sensor and madeit mobile at negative voltages.

Fig. 9 B displays currents associated with test depolar-izations from 150 to 20 mV (traces 3) or to 50 mV(traces 4) after conditioning pulses (Vcond) to variousvoltages. For test depolarizations to 20 mV (traces 3),calcium currents progressively declined with increasingVcond and attained a minimum at Vcond �40 mV. Atmore positive Vcond, calcium currents during the condi-tioning pulse were smaller and inactivated less (notshown); the reduced inactivation was evident in the in-creasing current magnitudes for test pulses at Vcond �40 mV. An opposite pattern was seen for charge move-ments recorded after test pulses to 50 mV (traces 4).At test voltages of 50 mV, ionic currents were verysmall and gating charge movements were easily resolv-able. Here, charge movements increased with Vcond to amaximum at 20–40 mV, and then declined at more pos-itive values. Importantly, blockade of the calcium cur-rents by gadolinium eliminated the effect of condition-ing (traces 5). The results provide a clear demonstra-tion that calcium current passing through the channelshifts the distribution of gating current transients tonegative potentials.

The pooled results from five similar experimentswith the �1733 mutant are shown in Fig. 10 A. There

was a bell-shaped voltage dependence for current inac-tivation during the conditioning pulse (circles), withmaximal inactivation occurring at 20–40 mV. The in-crease in gating charge movement following depolar-ization from 150 to 50 mV (filled bars) showed agenerally similar dependence on Vcond, with a maxi-mal value at Vconc 20 mV. When calcium currentswere blocked with gadolinium, intramembrane chargemovements were small and did not vary with Vcond (graybars). The correlation between ionic currents andcharge movement, and its dependence upon channelactivity, strongly support our conclusion that calciuminflux through the �1733 channels during the condi-tioning pulse promoted inactivating transitions thatboth reduced calcium currents and shifted the voltagedependence of intramembrane charge movements tonegative voltages. In all probability, these inactivatingtransitions are equivalent to those induced by increas-ing the cytosolic calcium concentration (Fig. 6).

Fig. 10 B displays the results of similar experimentsobtained with the wild-type channel. The bell-shapeddependence of current inactivation (circles) on Vcond

was essentially identical to that seen with the �1733 mu-tant. In contrast, however, there was no dependence ofcharge movement on Vcond in the wild-type channels(bars), indicating that were was no correlation betweenintramembrane charge movement and channel inacti-

Figure 9. Ca2� current promotes in-activated states of the voltage sensor inthe �1733 deletion mutant channels.(A) Pulse protocols used to monitoravailability of ionic currents (currenttrace 1, test pulse from 150 to 20 mV)and the intramembrane charge move-ment in inactivated channels (currenttrace 2, test pulse from 150 to 50mV). Conditioning pulses were from90 mV to Vcond. Pipette solution: 1EGTA. (B) Test currents recorded asshown in A. Conditioning voltages areindicated by the figures above thetraces. Traces 3 (test voltage 20 mV)and 4 (test voltage 150 mV) were re-corded with 0 Na bath solution. Traces5 (test voltage 150 mV) were re-corded in the same cell after the block-ade of ionic currents by Gd bathsolution.

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vation. The results are consistent with previous negativeobservations for the native channels. For the reasonsdescribed above, the negative observation does notnecessarily imply a lack of the interaction between cal-cium influx and the voltage sensors in wild-type cal-cium channels. Indeed, the presence of the effect inthe �1733 deletion suggests that the interaction doesexist in native L-type channels, but that the presence ofthe carboxyl terminus obscures its detection, perhapsby enhancing a subpopulation of channels that gatebut do not open.

D I S C U S S I O N

Inactivation of L-type channels has traditionally beensubdivided into voltage-dependent and calcium-depen-dent components. Here we propose that the voltage-and calcium-dependent components proceed by acommon mechanism, and that calcium governs therate at which inactivation occurs through its interactionwith the channel in the permeation pathway. The fol-lowing lines of evidence and comparisons with previouswork support this hypothesis.

Calcium Affects Voltage-dependent Gating

Direct perfusion of calcium into the cell through thepatch pipette, at concentrations �100 �M, producedcomplete blockade of ionic current, and this was asso-ciated with a large negative shift in the voltage depen-dence of charge movement in the OFF gating transi-tion. The shift has been previously associated withvoltage-dependent inactivation. The effects of intra-cellular calcium were reversed by NCX activity oper-ating in a calcium efflux mode (Fig. 3 A), and weremimicked by NCX operating in the calcium influxmode (Figs. 3 B and 4). Thus, channel inactivationinvolves the reversible interaction with high concen-trations of intracellular calcium (�100 �M); theseconcentrations are probably similar to the local cal-cium concentrations the channels experience duringactivity.

Both direct and reversed modes of NCX altered ionicand gating currents very rapidly in comparison to themuch slower response during direct perfusion with cal-cium. Even more surprising was the inability of 10 mMEGTA in the pipette solution to prevent the inhibitionof calcium current during reversed NCX activity. Thisimplies that NCX is very efficient in affecting calciumconcentrations local to the channel in this expressionsystem. Reverse NCX activity has previously been shownto load transfected CHO cells with as much as 6 mmolecalcium per liter cell water within 5 min (Reeves et al.,1996). In the present experiments, the effects of NCXactivity on the global cellular calcium concentrationare not known. Most likely standing concentration gra-dients of calcium are generated, reflecting the balancebetween NCX activity on the one hand and cellularperfusion on the other, with the steepest change in cal-cium occurring in the region beneath the plasma mem-brane, a region that would be directly sensed by the cal-cium channels. We have tried various combinations ofdifferent buffers, including BAPTA, to prevent the inhi-bition of calcium channels by reverse NCX activity; themost effective system was the use of the pipette solu-tions containing large (100 mM) quantities of glu-tamate or citrate, which are small, rapidly diffusing low-affinity calcium buffers.

Figure 10. Comparison of the reduction of calcium current andthe availability of charge movement from 150 to 50 mV afterconditioning at different voltages, measured as in Fig. 9. (A) Cur-rent inactivation (circles) and charge 2 (filled bars) in the �1733mutant. Current inactivation was measured as the reduction of themagnitude of test calcium current relative to the magnitude with-out conditioning (Vcond 90 mV), charge 2 was measured as thedifference between charge transfers at 50 mV obtained with andwithout (Vcond 90 mV) conditioning. Before averaging, the in-crease of the charge movement between 150 and 50 mV, wasrelated to the maximal charge transfer in each cell. The maximalcharge transfer was obtained in the Gd bath solution by pulsingfrom 150 to 60 mV. In the �1733 mutant, the charge movementrecorded in 0 Na solution (filled bars) was significantly increasedby conditioning pulses more positive than 20 mV (P � 0.05, un-paired Student’s t test). The charge movement in Gd solution(gray bars) was significantly increased only after 100 mV condi-tioning. (B) Current inactivation (circles) and charge 2 (bars) inwild-type channels. Current inactivation was similar to that in the�1733 mutant, but there was no significant change of chargemovement between 150 and 50 mV (0 Na bath solution, openbars).

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Calcium Accelerates the Changes of Gating CurrentsThat Were Previously Associated with Voltage-dependent Inactivation

The negative shift in the voltage dependence of the in-tramembrane charge movement is characteristic of cal-cium channels that have undergone voltage-dependentinactivation (Brum and Rios, 1987; Shirokov et al.,1998; Ferreira et al., 2003). In our experiments, in-creasing concentrations of intracellular calcium pro-gressively increased the rate at which the OFF charge at90 mV was reduced (Fig. 6). At 0.3 mM calcium in thepipette solution, the OFF charge declined nearly 100times faster (8 ms) than with 1 mM EGTA (0.7 s). Thesimplest interpretation of these results is that the rateof the inactivating transition induced by depolarizationincreases with the calcium concentration, i.e., thatthere is no mechanistic distinction between “voltage-dependent” and “calcium-dependent” inactivation un-der our experimental conditions.

Recent work of Ferreira et al. (2003) suggests that arapid component of voltage-dependent inactivation ofL-type calcium channels may reflect the charge immo-bilization mechanism. Within the limitations of ourability to define slow components of the relaxation ofthe charge movement transients, it appears that intra-cellular calcium did not promote charge immobiliza-tion. We did not detect a measurable slow componentof relaxation of the OFF transient, as it would be ex-pected for remobilization of the voltage sensor (e.g.,Fig. 7). This is also reflected in the double pulse exper-iments (Fig. 2 A), where complete decay of the OFFtransient occurred during the interpulse interval, andyet the subsequent ON transient during the secondpulse was dramatically reduced.

It is hypothetically possible that a shift in gatingmode, due to voltage- or calcium-dependent facilita-tion, could alter the voltage distribution of chargemovements. Voltage-dependent facilitation, however,would be minimized in our experiments through ouruse of the �2a splice variant (Cens et al., 1996; Qin etal., 1998). Moreover, calcium-dependent facilitationtypically occurs at much lower cytosolic calcium con-centrations (0.4 �M; Hirano and Hiraoka, 1994) thanthose required in our experiments to shift the voltage-distribution of the gating currents. Thus, we concludethat in our experimental conditions the effect of cyto-solic calcium on channel-gating currents was to acceler-ate the inactivating transitions conventionally associ-ated with voltage-dependent inactivation.

A minimal four-state diagram describes the charge 1/charge 2 interconversion mechanism as an allostericinteraction between two gates/processes (diagram 1;Brum and Rios, 1987). Voltage-dependent activatingtransition rest↔active and voltage-independent inacti-vating transition prime↔inactive are coupled accord-

ing to microscopic reversibility: KAP→AI · exp(V1/K) KRP→RI · exp(V2/K), where K is the slope factor, V1 andV2 are half-distribution voltages for charge 1 andcharge 2, respectively, and KAP→AI and KRP→RI are equi-librium constants. Results of our current work allowone to propose that the prime↔inactive transition ofthe scheme depends on intracellular calcium (diagram2). The coupling equation then becomes: (1/KDA) ·exp(V1/K) (1/KDR) · exp(V2/K), where KDR andKDA are dissociation constants for calcium in rest andactive states. Although the oversimplified diagram 2 ex-plains well why intracellular calcium affects the voltagesensor only after it moved into the active state: KDR KDA · exp((V1 V2)/K) � 500 · KDA, it clearly is notsufficient, since inactivation of the voltage sensor oc-curs even at very low (0.1 �M) intracellular calcium, yethigh concentrations of calcium (�10 �M) are requiredto speed it up. This problem is resolved by the eight-state model for three coupled processes illustrated inFig. S6, available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1.

Results of our work on the expressed cardiac L-typechannels are in accord with the findings of Leroy et al.(2002) on native cardiac L-type calcium channels. Inguinea-pig ventricular heart cells, applications of caf-feine promoted a negative shift of the voltage depen-dence of the intramembrane charge movement (Leroyet al., 2002). Since pretreatment with ryanodine and/or thapsigargin reduced the effect of caffeine on thecharge movement, the authors proposed that the caf-feine-induced interconversion between charge 1 and 2was mediated by calcium released from the sarcoplas-mic reticulum. Other studies of inactivation of L-typecalcium channels by calcium released from the sarco-plasmic reticulum (Sham et al., 1995; Adachi-Akahaneet al., 1996) provided strong evidence for functionalcoupling between L-type calcium channels and ryano-dine receptors in microdomains with restricted diffu-sion of calcium (for review see Rios and Stern, 1997).Our results indicate that the concentration of intracel-lular calcium has to be in the order of 10–100 �M to af-fect kinetics of inactivation, therefore providing inde-pendent, albeit indirect, support for the existence ofcalcium microdomains between the sarcolemma andthe sarcoplasmic reticulum.

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Calcium Interacts with Channels That Have Moved Their Voltage-dependent Gate

The simplest interpretation of how intracellular cal-cium affects gating currents is that calcium interactswith the channels after they have undergone voltage-dependent gating. The OFF transients, but not the ONtransients, were reduced, suggesting that the openingtransition of voltage sensors was required for calciumto affect gating. Calcium had no effect on charge move-ment at potentials more negative than 10 mV (Fig.5), a voltage at which about one-third of the chargemovement associated with channel opening has oc-curred. At more positive potentials, the reduction ofgating currents had a steep voltage dependence (Fig. 5D), indicating that the cooperative movement of volt-age sensors is required for calcium to interact with thechannel. In agreement with this interpretation, theOFF gating currents were reduced by calcium onlywhen the depolarizing pulse was long enough (�5 ms)for most of the gating current transient to be complete(see Fig. 7).

Taken together, these findings indicate that the cal-cium binding site responsible for inactivation is notavailable in the resting channel, and that calcium inter-acts with the channel only after it underwent a substan-tial voltage-driven transition leading to the opening.Such a transition may be the movement of the voltage-dependent gate. The site of interaction with calciummight therefore lie within the water-filled cavity be-tween the selectivity filter and the voltage-dependentgate (Doyle et al., 1998; Roux and MacKinnon, 1999).Alternatively, the site might be generated at a cytosoli-cally disposed location by a voltage-dependent confor-mational change of the S6 transmembrane segmentsthat precedes the final opening transition. Consistentwith either scenario, the S6 transmembrane segmentshave been shown to be important determinants of inac-tivation of various types of calcium channels (Zhang etal., 1994; Hockerman et al., 1995, 1997; Hering et al.,1996, 1997, 1998; Berjukow et al., 1999, 2000; Sokolovet al., 2000; Stotz et al., 2000; Stotz and Zamponi, 2001).Zong et al. (1994) previously proposed that Ca-depen-dent inactivation is closely linked with ion selectivity,implying an interaction of calcium with the channel’spermeation pathway

Calcium-calmodulin Binding to the Cytoplasmic Aminoand/or Carboxyl Termini Is Not Required forCalcium-dependent Inactivation

Inactivation of L-type channels depends on calcium/calmodulin binding to the amino terminus (Ivanina etal., 2000), and to the carboxyl terminus (Lee et al.,1999; Peterson et al., 1999; Qin et al., 1999; Zuhlke etal., 1999). Because intracellular calcium blocked cal-

cium currents and reduced OFF gating current tran-sients in the “IQ-AA” mutant (Fig. 8), in which neithermechanism would be applicable, we conclude that cal-cium/calmodulin-dependent regulation of inactivationis not necessary for calcium-dependent channel closureand the acceleration of inactivation at the level of gat-ing currents. How can this conclusion be reconciledwith the by now well-established role of calcium/cal-modulin in the calcium-dependent inactivation of ioniccurrents? Hypothetically, it is possible that the calmod-ulin-dependent mechanism, in contrast to the direct ef-fects of calcium, has no significant effect on intramem-brane charge movements. This seems unlikely, how-ever, in view of the parallel reduction in ionic currentsand charge movements documented in Figs. 2 and 10.

A more plausible, although speculative, possibility isthat calmodulin is required to maintain the inactivatingeffects of calcium during its permeation through theopen channel. Calcium ions passing through an openchannel are thought to affect the channel gating bypromoting occupancy of a closed state from which thechannel can reopen with a lower probability (Rose etal., 1992). The probability of reopening is reduced rap-idly, after the first opening in calcium, as comparedwith barium (Yue et al., 1990). This strongly suggeststhat calcium acts inside or near the permeation path-way and induces an inactivated state from which re-opening is blocked. With low cytosolic calcium concen-trations, local calcium gradients in the vicinity of an in-dividual channel would dissipate rapidly once channelclosure occurs. Assuming that inactivation requires thecontinued interaction of calcium with its inactivationsite, one can envision that modest conformationalchanges in the inactivated channel protein could leadto dissociation of the bound calcium and hence to theloss of the inactivated state. Calcium/calmodulin-medi-ated voltage-dependent interactions with the cytoplas-mic tails of the �1 subunit (Kobrinsky et al., 2003),might act as a “latch” to prevent such conformationalchanges and lock in the inactivated state of the chan-nel. According to this view, calcium/calmodulin wouldnot be required for inactivation induced by high cyto-solic calcium concentrations, as in our experiments, be-cause the inactivation site would be immediately reoc-cupied by calcium if dissociation occurred.

Calcium Influx Through the Open Channels Accelerates the Inactivating Transitions of the Voltage Sensors

In �1733 channels, calcium influx during conditioningdepolarizations induced additional inactivation and aproportional shift in the voltage distribution of in-tramembrane gating charge (Fig. 9). A similar observa-tion was made in the case of expressed N-type channels(Shirokov, 1999), but not for native L-type cardiacchannels (Shirokov et al., 1993). As discussed in re-

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569 Isaev et al.

sults, it is possible that calcium influx shifts the volt-age dependence of charge movement in wild-typechannels, but the effect is undetectable because mostwild-type channels gate but do not open. At the sametime, high cytosolic calcium affects movement of thevoltage sensor in most if not all wild-type channels. Thisimplies that the interaction does not require channelopening. This conclusion seems at odds with our find-ing, discussed above, that calcium interacts with thechannels only after the ON intramembrane chargemovements have been largely completed. Movement ofthe voltage-dependent gate, however, might not be suf-ficient to open the channel. The final closed-to-opentransitions are likely to have little intrinsic voltage de-pendence (Zheng and Sigworth, 1997, 1998; Sukharevaet al., 2003), and they may be blocked or inhibitied inthe nonconducting wild-type channels. Therefore, wepropose that both calcium incoming through the openchannels and elevated bulk calcium promote the samemolecular events by interacting with the inactivationsite, access to which is controlled by the voltage sensor.

Overview

Here we summarize our findings in the form of a work-ing hypothesis that provides a reasonable account forall the results presented here. We realize that there maybe alternate explanations for many of the individual el-ements discussed below and that charge immobiliza-tion may contribute to inactivation in some circum-stances. Nevertheless, the hypothesis provides a plausi-ble viewpoint for interpreting the data presented hereand for designing experiments in future studies. Chan-nel opening is thought to be a sequential process in-volving initial movements of the voltage sensors thatare linked to rearrangements of the S6 transmembranesegments, followed by additional transitions that resultin ionic conduction. These voltage-independent transi-tions, but not the preceding movements of the voltagesensors, appear to be blocked by interactions involvingthe carboxyl terminal segments of the �1 subunit inmost wild-type channels; hence, most channels gate butdo not conduct. The initial movements of the S6 seg-ments generate a calcium binding site which, when oc-cupied with calcium, blocks the subsequent voltage-independent transitions; hence, high calcium con-centrations block ionic current. The change in thechemical environment of the voltage sensor, as re-flected in the negative shift of the voltage dependenceof its movement, is preferentially associated with thechannel configuration stabilized by the binding of cal-cium to the inactivation site, accounting for the accel-eration of charge interconversion by calcium. In con-ducting channels, the high local calcium concentrationgenerated as a result of channel activity promotes chan-nel closure and the entry of the channel protein into

the same inactivated state described above—i.e., re-opening of the channel is prevented by calcium’s ef-fects on the voltage-independent transitions of the S6residues. Nonconducting wild-type channels obviouslycannot generate the local calcium gradients that wouldlead to charge interconversion. Because most wild-typechannels are nonconducting, little if any change of theintramembrane charge movement can be detected afterinactivation of ionic currents. Upon channel closure,the local calcium concentration gradient dissipates veryrapidly and this would lead to dissociation of calciumfrom the inactivation site and channel reopening. Theinteractions involving calcium/calmodulin at the IQmotif and other sites stabilize this configuration of thechannel and block or inhibit calcium dissociation fromthe inactivation site; hence, calcium/calmodulin accel-erates inactivation of calcium currents, but it is notnecessary when cytosolic calcium is directly elevated(by perfusion through the pipette or by reverse NCXactivity).

Further structure-function analysis is required in or-der to identify the inactivation site and to understandthe influence of bound calcium on voltage-dependentgating of these channels.

The authors would like to thank Molecular Resource Facility ofNJMS, Drs. J. Berlin, A. Harris, M. Nowycky, and N. Shirokova forcomments, and Dr. M. Condrescu for the NCX 1.1 clone.

The work was supported by NIH R01MH62838 to R. Shirokov.

Olaf S. Andersen served as editor.

Submitted: 23 May 2003Accepted: 16 March 2004

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