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Cell Calcium 42 (2007) 467–476

Calcium transporters and signalling in smooth muscles

Rachel Floyd, Susan Wray ∗

The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK

Received 5 April 2007; received in revised form 9 May 2007; accepted 14 May 2007Available online 10 July 2007

bstract

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), playcrucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle

ells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similarTP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several

soforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contributionf PMCA and the Na–Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has beguno elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth

uscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminala, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the

urface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae andholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the

ynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, closeo the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can beetter understood.

2007 Elsevier Ltd. All rights reserved.

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eywords: Contractility; Sarcoplasmic reticulum; Ca-ATPase; Uterus; Calc

. Introduction

Smooth muscle contractility depends upon regulatedhanges of intracellular [Ca]. These changes will occur onbackground of controlled low, resting intracellular Ca and

he subsequent need to lower Ca again following the rise of Caequired to stimulate contraction. Calcium transporters play aey role in all these processes, i.e. maintaining low resting Ca,levating Ca for contraction and restoring Ca for relaxation of

mooth muscle. In addition, by distributing Ca transporters inembrane microdomains or positioning them in close prox-

mity to cellular organelles, their effects can reach beyond

∗ Corresponding author at: Department of Physiology, The University ofiverpool, Liverpool L69 3BX, UK. Tel.: +44 151 794 5306;

ax: +44 151 794 5321.E-mail address: s.wray@liv.ac.uk (S. Wray).

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meostasis

a homeostasis. In this short review we will consider the rolend regulation of Ca transporters in smooth muscle, focussingn their effects on signalling and contractility. We considera efflux and influx across the plasma membrane, then Ca

elease and re-uptake across the sarcoplasmic reticulum (SR)embrane. We then examine interactions between the SR and

lasma membrane and lipid rafts.

. Plasmalemmal Ca fluxes

Calcium entry in visceral smooth muscle is controlled viahe membrane potential, as this determines the opening of

oltage-gated Ca channels [1,2]. L-type Ca channels (Cav1,igh voltage activated) sensitive to dihydropyridine are theain, and often only type of Ca channel expressed in many

mooth muscles [3,4]. These channels supply the bulk of Ca

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equired to activate the myofilaments and are therefore the tar-et of pharmaceutical interventions directed at for example,ypertension and premature delivery. Recently there has beenvidence provided to show expression of T-type (low voltagectivation) Ca channels, Cav3, in a variety of smooth muscles,.g. uterus [5], vas deferens [6], urethra [7], bladder [8], andascular tissue [9]. It is not yet clear what the functional rolef T-type Ca channels may be in smooth muscle, especiallyiven how hyperpolarised the membrane has to be for themo be activated, but it may be their expression increases withltered or pathological changes, e.g. pregnancy [10], over-ctive bladder [11], hypertension [12] and proliferation [9].a may also enter smooth muscle cells by non-specific cationhannels and receptor operated channels [3,4,13]. Leakagef Ca can also occur into the myocytes from the 1–2 mM Caresent in the extracellular fluid surrounding them.

Calcium entry therefore occurs down both electrical andhemical gradients and hence eliminates the requirement Caransporters and the expenditure of ATP [14,15]. However,here is still a fundamental requirement for control of mem-rane potential, channel opening and Ca efflux mechanismso restore cytoplasmic Ca concentrations after stimulations16].

Ca transporters lower Ca after it has risen and maintainow background Ca (nM) during resting conditions in smooth

uscle cells [17]. The two families of Ca transporter presentn the plasma membrane of smooth muscle cells are the Ca-TPase (PMCA) and Na/Ca exchanger. The contribution ofach of these transporters to efflux and their properties andegulation will be discussed in turn, with their functionalffects examined.

.1. The plasma membrane Ca-ATPase (PMCA)

The PMCA of smooth muscle and other cells is a P-ype Ca-ATPase regulated by calmodulin. The PMCA alsoounter-transports a proton, which may lead to pH changesround smooth muscle cells affecting PMCA activity and itsctivity in turn affecting intracellular pH [18,19]. When com-ared to the Na/Ca exchanger the PMCA has a relatively lowapacity for Ca transport. Four isoforms of PMCA (1–4) haveeen identified with 80–90% amino acid sequence homologynd 10 membrane-spanning regions. Sequence alignmentsuggest that the structure of the PMCA is similar to that of theR Ca-ATPase, with the latter differing primarily by havingmuch smaller COOH-terminal tail [20]. The COOH termi-us is responsible for the regulation of PMCA by calmodulin21]. The different isoforms of PMCA have different basalctivities, affinities for calmodulin and rates of activation andnactivation [22]. In smooth muscle, there is evidence forMCA activity to be regulated by cGMP and PKC as well asalmodulin. Indeed the regulation by Ca-calmodulin, which

inds to an autoinhibitory region of the pump and therebytimulating it, is reminiscent of activation of muscle myosinight chain kinase by the same Ca-calmodulin mechanism23]. As well as different tissue distributions there is also

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m 42 (2007) 467–476

dynamic regulation of their expression [24]. Collectivelyhese features suggest that the PMCA isoforms may haveifferent physiological roles. PMCA-1 has been described as‘house keeping’ Ca pump whereas PMCA-4 has the lowestasal activity and greatest stimulation by calmodulin and aole in shaping Ca signals has been suggested [25]. PMCA-

has been implicated in hearing loss and knock out miceor PMCA-2 have difficulty with balance [26]. PMCA-3 hashe most restricted distribution and has only been reportedn neuronal tissues. In smooth muscle there is good evidencehat PMCA-1 and 4 are expressed; indeed these two isoforms

ay be present in all cells [27,28]. In the uterus, expressionf PMCA-2b has also been reported [29]. When evaluatinghe roles of PMCA in Ca homeostasis and smooth muscleunction, their low level of expression and the lack of spe-ific inhibitors is problematic [30]. Eosin and carboxyeosinre selective, potent inhibitors of the PMCA more so thanhe Na/Ca exchanger [31] and have been used with reason-ble success to explore the importance of PMCAs in smoothuscle [32]. More recently genetic approaches of knock-

ut, knock-in and conditional targeting of PMCA have beenpplied [33–35].

There are suggestions in the literature that some iso-orms of PMCA may be localised to lipid rafts or caveolae;he sphingomyelin and cholesterol-rich regions of plasma

embrane. Indeed since the first examinations of caveolaeunction were made, a role in regulating Ca entry or exit wasonsidered [36,37]. In 1993 Fujimoto presented evidence thatMCA was enriched in caveolar regions of the membrane.owever little subsequent data have confirmed this, apart

rom evidence of PMCA-4 in cerebella synaptic membranes38], and it is unclear if PMCA is co-localised with caveolin-[39]. Therefore, while there is convincing evidence for a

aft localisation of the Na–Ca exchanger, it is questionablehether this is the case for PMCA in smooth muscle [40].

.2. Contribution of PMCA to Ca efflux

Studies of numerous smooth muscles have shown thatMCA is important for Ca homeostasis and function [41,42].n isolated uterine myocytes, 70% of Ca efflux occurred viahe PMCA during the decay of a calcium transient, with theemaining 30% removed by the Na–Ca exchanger [43]. Inhi-ition of both mechanisms prevented all calcium efflux. Thearcoplasmic reticulum was shown to play a role in regulatingfflux rates in uterine myocytes and will be discussed further.

Using PMCA-4 knock out mice we were able to determinehat 70% of calcium efflux could be attributed to the activityf this isoform [44]. In a study of intact rat uterus, it wasoncluded that 35% of SR released Ca (evoked by agonists)as extruded by the Na/Ca exchanger, and hence the remain-

ng 65% by PMCA [45]. The authors also concluded that the

oupling between SR Ca release and the Na/Ca exchangerhanged with gestation and altered its relative contribution toa efflux. Therefore in the uterus, irrespective of the sourcef Ca influx, PMCA plays the predominant role in Ca efflux.

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n colonic myocytes no role for Na/Ca exchange was founduring the decay of the Ca transient following depolarisation,lthough a role in extruding high lumenal SR Ca loads haseen proposed [46]. In ileum a role for the exchanger haseen shown [47].

Studies of bladder from genetically modified mice haveuggested that PMCA accounts for only 25% of relaxationhile the Na/Ca exchanger is responsible for the remaining5% [34]. The authors also explored the role of differentMCA isoforms in bladder Ca homeostasis [48]. They con-lude that PMCA-1 is necessary for Ca efflux and PMCA-4s essential for the Ca increase in response to agonist stimu-ated contraction. There is evidence that the different PMCAsoforms are activated by a sudden rise in Ca at differentates, with PMCA-3 being the fastest and 4 the slowest [26]lthough no direct evidence exists for this being the case inmooth muscle cells.

.3. Other roles for PMCA and summary

Roles for PMCA that extend beyond Ca signalling andontractility have been suggested as a result of studiesn transgenic animals. In vascular smooth muscle, abla-ion or knock down of PMCA-4 led to apoptosis [35,49].tudies have also shown that ischemia causes a loss ofMCA-1 in neuronal tissue, which can be mitigated byalpain [50]. These data point to the close associationetween PMCA activity, Ca homeostasis and cell physiol-gy/pathophysiology. Recently a novel function for PMCAn vascular smooth muscle has been proposed [33]. Theyuggest that PMCA-4b is complexed with neuronal NO syn-hase (n-NOS) and plays a role in regulating NO production,hereby influencing vascular tone independently of any directffect in lowering Ca. When PMCA-4b is over-expressed,ystolic blood pressure rose and the contractile response toigh-K depolarisations was enhanced compared to controlitter mates. It is hypothesised that this is because less NO isroduced, presumably due to a decrease in Ca around n-NOSnd a down-regulation of its activity. Mice that overexpressMCA-4b also show increased vascular reactivity [51]. There

s limited evidence that PMCA may be associated with vascu-ar smooth muscle proliferation [52] but while PMCA maye needed for good health, there is no evidence of a spe-ific disease resulting from changed function in any smoothuscle. However, as noted by Prasad et al. (2004) in vivo

tudies of knock out mice do not appear to have phenotypesorresponding to alterations in Ca signalling predicted fromn vitro studies, perhaps due to compensatory mechanisms.ndeed the in vitro studies would not have predicted the deaf-ess, cancer or infertility found in the mutant mice, revealingell type specific roles of the PMCA isoforms. In addition,he roles of the different isoforms and their splice variants

ay be more dynamic than earlier studies suggested [53].hus future work may shed more light on the role of PMCA

n smooth muscles and point to a role in their development,unction and pathophysiology.

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m 42 (2007) 467–476 469

.4. The sodium–calcium exchanger

The Na/Ca exchanger is a membrane spanning electro-enic antiporter, present in most cell types. It utilises the Naradient provided by the Na, K-ATPase, to drive the extrusionf Ca. During each cycle, a single Ca ion is exchanged forhree Na ions and thus it is electrogenic. The exchanger reac-ion may occur in either direction, based on the relative [Na].hus if extracellular Na is significantly reduced (30 mM) araded rise in [Ca]i occurs as the exchanger reverses and Canters the cell. Direct inhibition of the Na, K-ATPase withtrophanthidin, also causes a rise in [Ca]i in smooth muscle,hich the authors suggest is driven by a local rise in [Na]i and

ts subsequent effects on the Na/Ca exchanger [54]. Given thatuch effects have been shown in other smooth muscles in thebsence of a global rise in intracellular Na, local concentra-ion changes in functional microdomains within the plasma

embrane have been suggested to occur which may regulatehe exchanger and promote Ca entry [55]. During prolongedepression of the Na gradient it is possible to induce Canduced Ca release (CICR) from the SR [56]. In support of thea/Ca exchanger acting as a bidirectional exchanger, mice

hat overexpress NCX isoforms show an increased rate ofCa] decline during relaxation and an increase in Ca influx57].

The NCX is 938 amino acids in length and is thought toontain 9 transmembrane domains. The N-terminus is gly-osylated although this is not thought to be functionallyignificant for its physiological properties [58]. The NCXamily consists of 8 members, 5 of which encode mammalianxchangers, the other three are from C. elegans but sharenough sequence homology to fall into this family. NCX1–3ave been extensively studied in mammals and a number ofplice variants of NCX1 exist [59]. NCX1 is found ubiq-itously, with its expression being reported in a number ofmooth muscles in conjunction with either NCX2 as in thetomach [60] or NCX3 in pulmonary artery smooth muscleells [61]. Furthermore, as with PMCA, it is thought thatissue specific expression of splice variants of the NCX areailored to meet the metabolic requirements of specific organ62,63].

Extensive analysis of the localisation of the Na/Caxchanger has been performed over the last decade to deter-ine the possible molecular effects of reverse mode exchange

n a variety of cells. The Na/Ca exchanger has been showno reside in plasma membrane microdomains that overlie theR whereas PMCA may have a more uniform distribution64,65]. As a result of their distribution, it is thought that thewo proteins function in a compartmentalised fashion, wherehe PMCA regulates the low resting [Ca] in the bulk cytosolnd the Na/Ca exchanger regulates the small junctional spaceetween the plasma membrane and the SR.

More recently, it has been shown that the Na/Ca exchangers spatially coupled to the �2 isoform of the Na, K-ATPasen vascular smooth muscle, where the expression of �2 isirectly coupled to calcium signalling [66]. This link between

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he exchanger and the pump and Ca signalling is describedn more detail below.

The relative contributions of the Na/Ca exchanger and theMCA to calcium extrusion in smooth muscle have beeniscussed previously [42,45].

.5. The Na, K-ATPase

The maintenance of high intracellular potassium and lowodium concentration requires active transport of both ionscross the cell membrane via the Na, K-ATPase. As withMCA and the SR Ca-ATPase, the Na, K-ATPase is a P-

ype ATPase with 10 membrane spanning regions and shareslose homology with the SR Ca-ATPase; more so than theMCA [67]. Although the Na, K-ATPase has been classicallyescribed as having 2 domains, � and �, a third transmem-rane accessory protein has been identified and classified as� subunit and is a member of a large family of single-spanembrane proteins, some of which functionally interact with

he Na, K-ATPase called the FXYD proteins. Expression ofa, K-ATPase isoforms is species and tissue dependent and

ach has differing kinetics for ion transport, Ca sensitivity

nd ouabain affinity. Specific regulation of Na, K-ATPase �soform expression has been shown in a number of smooth

uscles including uterus [68–70], small intestine [71], lung72] and bladder [73].

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ig. 1. Synoptic view of the primary mediators of calcium homeostasis in smoothnd cholesterol-rich membrane domains with Na, K-ATPase �2 isoforms (NKA�2)CaM) and Na, K-ATPase �1 isoforms (NKA�1) and sarcoplasmic/endoplasmicomplexes.

m 42 (2007) 467–476

More recently, it has become clear that each of the �soforms has a specific physiological role [74]. An emerg-ng view in vascular smooth muscles and other cells ishat the �2 isoform may directly regulate calcium trans-ort, since spatial coupling of the Na/Ca exchanger with the2 isoform of Na, K-ATPase can regulate [Na] in a sub-arcolemmal domain adjacent to SR Ca release sites [75].eterozygous �2+/− mice demonstrate increased myogenic

one and elevated blood pressure; similar changes also occurith application of nanomolar ouabain in wild-type animals

76]. Thus there is emerging evidence that �2 is specifi-ally coupled to activation-relaxation pathways in vascularmooth muscle. Further confirmation of the direct link thatxists between �2 and calcium homeostasis can be seenn other �2+/− tissues. Heterozygous �2 hearts are hyper-ontractile which the authors suggest may be mediated byncreased calcium released from the SR during contraction77]. Perinatal diaphragms from these mice show relaxationhat is 1.7-fold faster than wild-type, with �2 and Na/Caxchanger expression that is co-regulated. Furthermore, nossociated compensatorary changes in other proteins that reg-late calcium homeostasis such as the SERCA, PMCA or

hospholamban were reported [78] (Fig. 1).

Endogenous cardiac glycosides are now considered toediate blood pressure by indirect interaction with the Na/Ca

xchanger due to inhibition of the sodium pump. This leads

muscle. Sodium–calcium exchangers (NCX) co-localised within caveolae, plasma membrane calcium ATPases (PMCA) with associated calmodulinreticulum Ca-ATPases (SERCA) with associated phospholamban (PLN)

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o an increase in cytoplasmic Na causing Ca influx, whichesults in sustained contraction, an effect shown in vascularmooth muscle cells [79–81]. The literature surrounding thexistence of endogenous cardiac glycosides is as varied andontroversial as the compound name itself. Initially believedo be an isomer of ouabain, endogenous ouabain is now under-tood to be a circulating inhibitory factor [82]. Endogenousuabains are synthesized in the adrenal cortex [83] and mayerve as natural ligands for the cardiotonic steroid bindingite on the �2 and �3. Endogenous ouabain is locally pro-uced in other tissues such as the hypothalamus [84,85] andt has been suggested that ouabain may act as a natriureteicormone [79,86] with a vasoconstrictive effect [87].

The co-localisation of the �2 isoform of the Na, K-ATPaseith the Na/Ca exchanger in regions in close proximity to

he underlying SR has offered some support to the pro-osed mechanism of the endogenous ouabain action [88]. Its thought that binding of the inhibitor to the � subunit of thea, K-ATPase prevents active transport and causes an incre-ental, local rise in Na which forces the Na/Ca exchanger

nto calcium entry mode. Exactly what occurs next is stillubject to speculation although studies have shown that inardiac cells local increases in calcium directly affect con-ractility, thus exerting a positive inotropic effect [59]. Littles known of the cascade of events that occurs in smooth mus-le during ouabain inhibition. However as the SR exerts aegative feedback on contractility in several smooth muscles89–91], it would be expected that the effects would differrom cardiac muscle, where the SR is directly linked to thetrength of contraction. It is likely that in smooth muscle, thebserved decrease in membrane potential negativity associ-ted with ouabain exposure [92] could initiate an increasen contractile activity through voltage mediated effects, i.e.-type Ca entry, rather than reversal of the Na/Ca exchanger.

Further functional synergy between the Na, K-ATPase andhe Na/Ca exchanger is found in their co-regulation by phosp-olemman, also referred to as FXYD1 [93]. Overexpressionf FXYD1 in rat cardiac myocytes causes contraction andCa]i transient abnormalities similar to those shown in NCX1nockdown studies [94,95]. Functional studies have shownhat the cytoplasmic tail of FXYD1 directly interacts withhe intracellular loop of NCX1 causing inhibition [96] andhosphorylation of serine 68 of FXYD1 may be involved inAMP-dependent regulation of smooth muscle force [97].XYD1 is a known substrate for several kinases includingKA and PKC and studies suggest it is either itself an ionhannel or a regulatory subunit of one [98].

. SR Ca release and uptake

.1. Introduction

Stimulation by agonists leads to IP3 and diaclyglycerolroduction in the smooth muscle plasma membrane. Theajor effect of IP3 within the cell is to trigger Ca release

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rom the SR via IP3 receptors (IP3R) [99,100]. Ca itself canlso bind to ryanodine receptors (RyR) on the SR causinga release. Thus the intracellular Ca store in smooth musclelays an important role in elevating cytoplasmic Ca duringtimulation [101]. The ability of the SR to contain a higherhan cytosolic [Ca] requires active uptake of Ca into the SRnd this is accomplished via a Ca-ATPase.

Recent visualization of smooth muscle SR in living cellssing fluorescent labels tagged to thapsigargin (an inhibitor ofhe SR Ca-ATPase) or ryanodine, has shown the well devel-ped reticulum that is present [102]. Both the Ca-ATPasend RyR were distributed in a non-homogenous manner inreshly isolated uterine myocytes. Others have reported thatP3R in smooth muscle cells are also adjacent to caveolae onhe plasma membrane [103]. Furthermore there was clearly alose proximity of the Ca pump and RyR, to the plasma mem-rane. In the uterus and other tissues the SR also is profuseround the nucleus.

When the Ca store in the SR is visualized using low affinitya indicators such as mag-fluo-4, a rich reticular formationan be seen with the high Ca sites distributed in a simi-ar fashion to the Ca-ATPase. Spiral-shaped tubes appearo run along the longitudinal axis of uterine and vascular

yocytes [102,104]. Previous electron micrograph studiesad also indicated a well developed SR in smooth muscle anduggested it occupied from 1.5 to 7.5% of the cell volume.

.2. SERCA—the SR Ca-ATPase

It is well established that active transport is required tomport Ca into the lumen of the SR. The Ca transportingTPase located in the SR membrane that drives this transport

s known as SERCA. SERCA is another P-type ATPase, inommon with the plasma membrane Na and Ca-ATPases. Toate three genes have been identified which code for SERCAumps; SERCA 1, SERCA 2 and SERCA 3. These genesay also be spliced to give further variety to the pump. The

D structure of SERCA 1 in Ca bound and inbound formas published in 2002 [105], allowing detailed information

bout the structural changes in SERCA that occur with Caransport, to be obtained [106]. Studies suggest that two Caons from the cytoplasm bind and the pump is subsequentlyhosphorylated by ATP at Asp351. This high energy, phos-horylated intermediary translocates the Ca ions into the SRumen and in doing so becomes a low energy intermediary.here is also the counter transport of two or possibly threerotons ensuring partial charge balancing [107]. As notedarlier, protons are also translocated during the operation ofhe plasma membrane Ca-ATPase. The protons are releasednto the cytoplasm after two Ca ions bind, and the cycle isomplete.

In smooth muscle SERCA 2b, which encodes for a

10 kDa protein has been identified but this appears toe expressed in all tissues whereas SERCA 2a, which isostly found in cardiac muscle, has been reported in uter-

ne smooth muscle [108]. Changes in the two isoforms

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ave been reported to occur in human myometrium aroundabour [109]. This suggests that smooth muscles are able tohift SERCA isoform expression to meet changing phys-ological function, but this has not been investigated inetail.

.3. SERCA and smooth muscle relaxation

While the necessity to release Ca from the SR lumenaltore in smooth muscle to augment or modify Ca signallingas been reasonably well studied, less is known about theontribution of SERCA to lower cytosolic Ca and terminatea signalling. Evidence has however recently accumulatedointing to such a role for the SR Ca pump. By making directeasurements of lumenal Ca in uterine SR, we were able

o show uptake of Ca into the SR when cytosolic Ca wasigh, e.g. following depolarisation [110]. Interestingly, over-oading the uterine SR with Ca did not affect IP3-induceda signalling, but lowering the lumenal Ca content substan-

ially reduced it [110]. The SR’s ability to store Ca, i.e. acts a sink, is of course finite, and it may be that this uptakes coupled to a vectorial release to the plasma membrane Caxtrusion mechanisms. There is good evidence for this in uter-ne myocytes, where the SR was shown to be necessary for

aximal functioning of the Ca extrusion mechanisms [43].hus although the SR Ca-ATPase acting alone, i.e. with extru-ions mechanisms blocked, could not lower cytosolic Ca2+,hen functioning the SR Ca-ATPase augmented Ca efflux.nloading of lumenal SR Ca may by-pass the bulk cytoplasm

nd take place in the sub-plasma membrane microdomain,ften referred to as the superficial barrier [111]. Interactionsetween the SR and plasma membrane are discussed furthern the next section.

The sites of SR Ca release and re-uptake on the SR mem-rane in smooth muscle appear to be spatially separated112]. We propose that the agonist releasable part of theR is, under physiological conditions, full and primed forelease but that there may be another Ca pool that is involvedn the vectorial release of Ca to the plasma membrane effluxites [102]. In another study of uterine myocytes, Ca ‘hotpots’, i.e. a non-homogenous distribution of Ca, as reportedy Fluo-3FF (a low affinity Ca indicator), were found [113].hese data are also compatible with Ca release and re-uptakeccurring at different sites on the SR membrane. Others haveroposed morphologically separate Ca stores within individ-al smooth muscle stores [114].

. SR–plasma membrane calcium interactions

In many smooth muscles, sphingolipid and cholesterol-ich domains known as rafts, or caveolae when incorporating

he protein caveolin, are found in close proximity to theeripheral SR [103]. As discussed previously, the SR canake surface couplings with the plasma membrane but they

o not seem to join at any point to the adjacent caveolae.

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uch spatial coupling within a microdomain may be benefi-ial in maintaining tight regulation of calcium homeostasisnd signalling in smooth muscle [115–117].

Caveolae have been identified as congregation sites forultiple proteins involved in calcium signalling in smoothuscle [118,119]. Caveolae are usually 50–90 nm in size

nd increase the surface area of the cell significantly (∼70%).hey can also couple numerous signalling proteins together insingle restricted area, sometimes referred to as a signalome

120]. Among the most studied of signalling molecules arerc, Ras and EGFR (epidermal growth-factor receptor) whichre all functionally coupled within caveolae [121]. Recenttudies have shown that in ureter [122] and both non-pregnantnd late pregnant human myometrium, Ca-activated K (BK)hannels associate with and are localised in caveolae con-aining caveolins 1, 2 and 3 [123]. There is also evidenceor other ion channel subunits being preferentially located inipid rafts [124]. Recent work has shown BK channel internal-sation with lipid raft disruption [125]. Microdomains haveeen shown to facilitate the close regulation of Ca releaserom the SR store, refilling of the store through plasma mem-rane store-operated channels [126] and influence both theytoplasmic [Ca] and SR [Ca] through modulation of thea/Ca exchanger [88].Evidence shows that the Na/Ca exchanger is enriched in

aveolae in smooth [127] and cardiac muscle [128]. Theres mounting evidence to suggest that the Na/Ca exchangernd Na, K-ATPase �2 are spatially and functionally cou-led in a variety of cells [129]. Direct measurement ofocalised calcium signalling using the calcium sensor pro-ein GCaMP2 tethered to Na, K-ATPase �2, has confirmedpecific localisation to PM microdomains overlying the SRnd also demonstrated store-operated Ca entry in arterialmooth muscle cells [130]. Recent studies have shown thatlarge proportion of total cellular Na, K-ATPase residesithin caveolar domains and can act as a signal trans-ucer in vascular smooth muscle cells [131,132]. Exposureo cardiac glycosides causes the Na, K-ATPase to interactith neighbouring membrane proteins to relay messagesia intracellular signalling complexes to mitochondria andhe nucleus [133]. The most frequently cited signallingascades that are initiated by Na, K-ATPase interactionith ouabain are Src activation and EGFR transactiva-

ion, PKC activation and the Ras/Raf/MEK/ERK1/2 cascade134,135].

Thus the pathways for interactions between the SR andlasma membrane are numerous in smooth muscle. Mem-rane microdomains are the focus of much current researchnd their influence on Ca transporters may soon be under-tood more clearly.

. Conclusions

In summary, Ca transporting mechanisms play a vital rolen all types of smooth muscle. These Ca pumps establish

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nd maintain the necessary Ca gradients between the plasmaembrane and cytoplasm and the cytoplasm and sarcoplas-ic reticulum, to ensure that the Ca signals necessary for

xcitation and contraction occur in smooth muscle cells.everal genes and splice variants exist for the Ca-ATPasexpressed in smooth muscle. With new data provided byrystal structure analysis and transgenic animals, along withodern imaging techniques and pharmacological tools, good

rogress is being made in understanding the function and reg-lation of these transporters. Information has to be integratedo that their roles in intact, physiological conditions, whichverlap with exchangers and ion channels, can be better eluci-ated. An area of current research focuses on how membraneicrodomains and caveolae dynamically affect transporter

ctivity, and the role of sub-plasmalemmal spaces, especiallyetween the SR and caveolae, in shaping Ca signals and otherignalling cascades. Although not the focus of this review,he clear link between metabolism and energy-dependenta-ATPases makes it clear that they will also contribute to

mooth muscle pathologies caused by ischaemic or hypoxiconditions.

cknowledgements

We are grateful to Michelle Nelson for excellent secre-arial assistance in the production of this article and past andresent laboratory members who have contributed to the workescribed.

Supported by Mersey Kidney Research, Action Medicalesearch and Wellbeing of Women.

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