CRAC channels: activation, permeation, and the search for a molecular identity

Cell Calcium 33 (2003) 311–321

CRAC channels: activation, permeation, and the searchfor a molecular identity

Murali Prakriya, Richard S. Lewis∗Department of Molecular and Cellular Physiology, Stanford University School of Medicine,

Beckman Center B-111A, Stanford, CA 94305, USA

Received 5 February 2003; accepted 10 February 2003

Abstract

The Ca2+ release-activated Ca2+ (CRAC) channel is a highly Ca2+-selective store-operated channel that is expressed in T lymphocytes,mast cells, and other hematopoietic cells. In T cells, CRAC channels are essential for generating the prolonged intracellular Ca2+ ([Ca2+]i )elevation required for the expression of T-cell activation genes. Here we review recent work addressing CRAC channel regulation, poreproperties, and the search for CRAC channel genes. Of the current models for CRAC current (ICRAC) activation, several new studiesargue against a conformational coupling mechanism in which IP3 receptors communicate store depletion to CRAC channels throughdirect physical interaction. The study of CRAC channels has been complicated by the fact that they lose activity in the absence ofextracellular Ca2+. Attempts to maintain current size by removing intracellular Mg2+ have been found to unmask Mg2+-inhibited cation(MIC/MagNuM/TRPM7) channels, which have been mistaken in several studies for the CRAC channel. Recent studies under conditionsthat prevent MIC activation reveal that CRAC channels use high-affinity binding of Ca2+ in the pore to achieve high Ca2+ selectivity buthave a surprisingly low conductance for both Ca2+ (∼10 fS) and Na+ (∼0.2 pS). Pore properties provide a unique fingerprint that providesa stringent test for potential CRAC channel genes and suggest models for the ion selectivity mechanism.© 2003 Elsevier Science Ltd. All rights reserved.

Keywords: CRAC channel; CRAC current; T cells; Store-operated channels; TRP channels

A widespread mechanism for calcium signaling occursthrough the opening of plasma-membrane Ca2+ channelsin response to the emptying of intracellular Ca2+ stores.This so-called capacitative, or store-operated, Ca2+ entrypathway is practically universal among non-excitable, andprobably many types of excitable cells[1]. Electrophys-iological studies have revealed a wealth of informationabout the diverse characteristics of store-operated channels(SOCs). From these studies it has become apparent thatSOCs comprise an extended family whose members aredistinguished by different degrees of Ca2+ selectivity, rang-ing from non-selective to among the most Ca2+-selectivechannels known[2,3]. The molecular composition of thesechannels, as well as the mechanism that links store deple-tion to channel activation is still a major unsolved mysteryin most cells.

The SOCs of T lymphocytes and mast cells were the firstto be studied with patch-clamp techniques and have been themost extensively described from a biophysical standpoint.

∗ Corresponding author. Tel.:+1-650-723-9615; fax:+1-650-725-8021.E-mail address: [email protected] (R.S. Lewis).

This subset of SOCs, known as Ca2+ release-activated Ca2+(CRAC) channels, are distinguished from other SOCs bytheir extremely high selectivity for Ca2+ and extremely lowconductance[2,3]. In this review we will first outline thereasons why the CRAC channel is a particularly attractivetarget for research within the SOC channel family, and thendiscuss recent progress in understanding its regulation andmolecular basis.

1. The CRAC channel as a target for SOC research

1.1. The CRAC channel is a prototypic store-dependentchannel

Activation of cell surface receptors can elicit Ca2+entry through several classes of ion channels, includingligand-gated, second-messenger-operated, and store-operatedsubtypes. SOCs are most strictly defined as being activat-able by a decrease in the lumenal concentration of Ca2+in stores, independently of receptor stimulation. In T cellsand mast cells, CRAC channels are activated by antigen

0143-4160/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0143-4160(03)00045-9

312 M. Prakriya, R.S. Lewis / Cell Calcium 33 (2003) 311–321

receptor stimulation that triggers Ca2+ release from theER [4–9]. While they may receive convergent inputs frommultiple pathways, CRAC channels are considered to beprototypic SOCs based on strong evidence that the channelresponds to store depletion per se rather than to Ca2+ re-leased from stores, or to various other signaling pathwaysactivated through PLC-coupled receptors (e.g. G proteins,diacylglycerol, etc.).

CRAC current (ICRAC) is also activated by SERCA in-hibitors like thapsigargin (TG), Ca2+ ionophores like ion-omycin, and intracellular Ca2+ chelators (EGTA, BAPTA),agents that interfere with Ca2+ store loading and cause pas-sive depletion (see[2,3] for review). It is important to notethat these approaches by themselves do not prove store de-pendence: TG and ionomycin generally cause a rise in intra-cellular Ca2+ ([Ca2+]i ), which can activate Ca2+-sensitivechannels, and chelators can in principle activate channels byremoving inhibitory actions of resting Ca2+. Thus, it is sig-nificant thatICRAC can be activated by depleting agents likeTG or ionomycin even when [Ca2+]i is highly buffered toa constant value, demonstrating that changes in [Ca2+]i arenot necessary for activation[10,11]. Furthermore, the abil-ity of TPEN, a permeant chelator that reduces [Ca2+]i inthe ER, to reversibly activateICRAC in RBL cells demon-strates that store depletion rather than Ca2+ release per seis the relevant stimulus (suggesting that a more apt namefor the channel may have been “depletion-activated”)[12].The precise mechanism by which store depletion is linkedto channel activation is still obscure (see below).

The capacitative Ca2+ entry hypothesis as originallyproposed by Putney states that SOCs are activated byIP3-mobilizing receptors solely through their ability to de-plete Ca2+ stores[13]. This idea has been challenged inrecent years by two sets of observations. The first is that anumber of presumed SOCs (Imin, TRPC1, TRPC3) appear torequire IP3 receptors and IP3 for activity [14–17]. Anotheris the recent finding that in DT40 cells in which PLC� hasbeen knocked out, a muscarinic agonist releases Ca2+ fromstores without activating Ca2+ influx. Surprisingly, however,in the same PLC�−/− cells store depletion by TG is able toevoke Ca2+ entry [18]. These results raise the question ofwhether store depletion is the actual physiological stimulusthat opens SOC channels in intact cells, and in particu-lar whether multiple signaling pathways (IP3, PLC, storedepletion, etc.) must converge on the channel to activate it.

To our knowledge there is no published instance of mem-brane receptors activating CRAC channels without alsodepleting stores. Thus, the simplest interpretation of theavailable data is that store depletion is necessary, althoughit may not in all cases be sufficient, to activateICRAC. Theobservation that a muscarinic agonist can release Ca2+ fromstores without activating Ca2+ influx in the PLC�−/− DT40cells raises a number of interesting possibilities. First, PLC�may be involved in a structural capacity to maximize the ef-ficiency of IP3-generation to deplete Ca2+ stores that in turnactivateICRAC. Several studies suggest that a subset of stores,

perhaps close to the plasma membrane, may be preferentiallycoupled to CRAC activation[19,20] (for review, see[21]).Perhaps PLC� functions to localize the “CRAC stores” closeto PLC-coupled receptors[22]. Another possibility is thatreceptor activation in the absence of PLC� produces a signalthat inhibits CRAC activity. Further studies of the identityand location of these stores, as well as the binding partnersand activities of PLC�, will help to clarify these models.

1.2. The CRAC channel is essential for geneexpression in activated T cells

Under physiological conditions, CRAC channels in T cellsopen in response to the recognition of antigen by the T-cellreceptor (TCR). Antigen binding leads to activation of PLC�and generation of IP3, which releases Ca2+ from the ER andthereby activatesICRAC [7]. It is well established throughpatch-clamp recordings that the biophysical and pharmaco-logical properties of the currents activated in T cells by anti-gen receptor crosslinking, IP3, intracellular Ca2+ chelators,ionomycin and TG are identical[4,5,10]. Moreover, mutantT cells selected for the absence of CRAC channel activ-ity lack antigen receptor-mediated Ca2+ entry [23]. Thus,CRAC channels appear to be the only Ca2+ influx pathwaythat is engaged by the TCR.

The loss of CRAC channel function leads to a devastatingloss of immune function in vivo. In two studies of human pa-tients, severe combined immunodeficiency (SCID) has beentraced to an inability of store depletion to activate Ca2+ in-flux through CRAC channels[8,24]. The great majority ofCa2+-dependent genes that are activated in response to stim-ulation of the TCR fail to be expressed in these patients eventhough artificial elevation of [Ca2+]i can rescue the defect[24]. Many of these defects arise from an inability of thecells to activate NFAT[25], an essential transcription fac-tor that requires prolonged [Ca2+]i elevation for activation[26,27].

The critical physiological roles of CRAC channels in Tcells and their clear dependence on the content of intracel-lular Ca2+ stores makes the CRAC channel a particularlyattractive target for SOC channel research. Thus, under-standing the regulation and identity of the CRAC channelat a molecular level is an important long-term goal. Be-low we review recent studies that have addressed thesequestions.

2. Regulation of CRAC channels by store depletion

Despite a great deal of work published over the past 10years, the nature of the signal that links store depletionto activation of CRAC channels is still unclear. The mostwell-known hypotheses currently include a diffusible activa-tor that is synthesized and/or released from the ER followingstore depletion, the insertion of active CRAC channels intothe plasma membrane through a vesicle fusion mechanism,

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and functional coupling between CRAC channels and pro-teins in the ER membrane. It is fair to say at this pointthat none of these mechanisms has yet received widespreadsupport, nor have any of them been definitively ruled out.These issues have been the subject of several recent reviews[2,3,21,28–30]. Surprisingly, even the most basic parame-ter of SOC activation, the dependence on lumenal [Ca2+]in the ER, has never been quantified, and evidence existsfor a graded as well as a highly cooperative, threshold-likeresponse[12,20,31].

2.1. Recent tests of the conformational couplingmodel of CRAC activation

Perhaps the most popular hypothesis to explain the ac-tivation of SOCs is the “conformational coupling” model,originally based by analogy on the functional coupling ofvoltage-gated Ca2+ (CaV) channels and ryanodine recep-tors in skeletal muscle[21,32]. According to this idea, IP3receptors in the ER are physically linked to CRAC chan-nels. Depletion of stores changes the conformation of theIP3 receptor, and this is transmitted to the CRAC channel,causing it to open. A corollary of this model is that Ca2+entry following receptor stimulation involves a close phys-ical interaction between the endoplasmic reticulum, whereIP3 receptors are localized, and the plasma membrane. This

Fig. 1. Effects of 2-APB onICRAC. Plot of peakICRAC during pulses to−100 mV before and after exposure of a TG-pretreated Jurkat cell to 40�M2-APB (bar). Application of 2-APB results in a transient enhancement ofICRAC which is followed by poorly reversible inhibition. In parallel with currentinhibition, this high concentration of 2-APB also removes fast inactivation ofICRAC. Examples of currents at various time points are shown below toillustrate the removal of fast inactivation. Adapted from[42].

idea was supported by evidence that in some cells, forma-tion of a cortical actin band can interrupt Ca2+ influx, as ifthe ER is prevented from making contact with the plasmamembrane[33,34]. Additional evidence came from reportsof binding of IP3 receptors to some members of the TRPchannel family (TRPC1 and TRPC3) that may operate asSOCs, and from effects on TRP activity of IP3 receptorsand fragments thereof[14,15,17,35,36]. However, ICRACin RBL cells is not affected by actin stabilization or de-polymerization[37], and it is uncertain to what extent theactivation mechanism of TRPC1 and TRPC3 is shared withendogenous SOCs, including the CRAC channel[38].

Acceptance of the IP3R-based conformational cou-pling model increased following reports that 2-APB, amembrane-permeant antagonist of IP3 receptor function[39], inhibits TG-stimulated Ca2+ influx in several cells[34]. Inhibition of store-operated Ca2+ entry by 2-APB wastaken as evidence for a role of IP3 receptors in the activationof SOCs and therefore as proof for the conformational cou-pling mechanism of activation of SOCs[34,40]. Subsequentelectrophysiological studies confirmed that 2-APB does in-hibit ICRAC in Jurkat T cells and RBL cells[37,41–43]. Inaddition, 2-APB was found to reduce fast inactivation ofICRAC in parallel with the slow development of its inhibitoryeffect [42] (Fig. 1). Fast inactivation of CRAC channelsoccurs over tens of milliseconds during hyperpolarizing


voltage steps and results from feedback inhibition by Ca2+at sites thought to lie several nanometers from the channelpore [44]. Interestingly, an early hypothesis suggested thatfast inactivation of CRAC channels occurs through bindingof Ca2+ to CRAC-coupled IP3R and thus that 2-APB mightinfluence fast inactivation by inhibiting with the IP3R [21].

Despite the strong appeal of the conformational couplingidea, many lines of evidence indicate that this model, atleast as it applies to coupling between IP3R and CRAC, isincorrect. Sugawara et al. first showed that in a DT40 cellline in which all three isoforms of the IP3R were genet-ically deleted, TG-induced Ca2+ influx was normal[45].Subsequent patch-clamp experiments showed that these cellsexpressICRAC at normal levels, and fast inactivation alsooccurred normally[42]. Furthermore, heparin, a competitiveantagonist of IP3Rs, fails to inhibit activation or the main-tenance ofICRAC by store depletion[42,46–48]. In contrast,currents mediated by recombinant TRPC3 channels in ex-cised patches from HEK293 cells or endogenousImin chan-nels from A431 cells require the presence of IP3 to maintainactivity and are potently blocked by heparin[15,16]. Thus,CRAC channels differ significantly from these other chan-nels in requiring neither IP3 receptors nor even backgroundlevels of IP3 for activation.

Given that IP3Rs are not required forICRAC activation,how can the effects of 2-APB be explained? Interestingly,just as in wild-type DT40 cells, 2-APB elicits the same com-bination ofICRAC inhibition and removal of fast inactivationin IP3R-knockout cells[42]. Thus, the multiple actions of2-APB onICRAC are unrelated to IP3 receptors. The site orsites of action underlying these effects are unknown. 2-APBis much less effective at inhibitingICRAC when applied intra-cellularly rather than extracellularly[41–43], suggesting thatthe site of action is not intracellular. However, this is not nec-essarily strong proof if 2-APB is sufficiently hydrophobic topass freely across membranes. Under such conditions, when2-APB is applied through the patch pipette, the concentrationat the membrane will be reduced because the rate-limitingstep for diffusion is across the pipette tip rather thanacross the membrane. However, acidification of the bathingmedium, which would be expected to increase protonation ofextracellular 2-APB and reduce its membrane solubility doesnot inhibit its effects, consistent with an extracellular site ofaction[42]. On the other hand, several studies of TRP chan-nels (TRPV6/CaT1, TRPC3) have suggested that 2-APBinhibits SOCs by interfering with depletion–activationcoupling [49–51], which is presumably intracellular. Onecaveat in the use of 2-APB to study the activation mecha-nism is that the drug is known to affect other ion transportproteins not obviously related to CRAC channel activation,including the mitochondrial Ca2+ release machinery[42],voltage-dependent K+ channels[52], and SERCA Ca2+pumps[53]. Certainly, the effects of 2-APB on a process asmysterious as SOC activation must be interpreted cautiously.

Despite the many problems associated with 2-APB, anintriguing feature of this drug is its ability to not only in-

hibit CRAC channels, but also to enhance their activity inresponse to store depletion[42,50]. The stimulatory effect ismost clearly seen at concentrations in the range of 1–5�M,where no inhibition is observed. At these dilute concentra-tions, 2-APB enhances Ca2+ entry in intact cells and in-creasesICRAC amplitude by up to five-fold[42]. To ourknowledge, this is the first example of a pharmacologicalagent that can enhance the activation ofICRAC beyond thelevel evoked by complete store depletion. Potentiation ofCRAC channels also does not require IP3Rs [42,50]. Nei-ther the locus of the 2-APB effects nor its mechanism ininducing potentiation and depression of CRAC currents arecurrently known.

In sum, CRAC channels appear not to be activated througha conformational coupling mechanism that involves physicalinteraction with IP3 receptors in the ER membrane. How-ever, these results do not rule out conformational couplinginvolving other ER proteins, such as the ryanodine receptor,which have been proposed to mediate activation of SOCs.Ryanodine receptors have been reported to interact func-tionally with TRPC3[54], and pharmacological inhibitorsof RyRs (ruthenium red, 8-N-cADPR, ryanodine) interferewith TG-activated Ca2+ influx in IP3R−/− DT40 cells[55].Based on these results, it has been suggested that CRACchannels can engage in conformational coupling with RyR,and the presence of normalICRAC in IP3R−/− DT40 cells isdue to compensatory coupling to RyRs[55]. However, thereare several inconsistencies with this idea. First, rutheniumred suppressesICRAC to the same degree in both IP3R−/−and wt DT40 cells, whereas a greater suppression would beexpected in IP3R−/− cells, in which all coupling was throughRyR. Second, acute introduction of heparin intracellularly,which would be expected to block effects of IP3R beforeRyRs have had a chance to compensate, does not suppressICRAC [42,46–48]. Finally, as with 2-APB, it is importantto rule out direct inhibitory effects onICRAC. There is nostrong reason to suppose that activation of SOCs by con-formational coupling must involve Ca2+ release channelsrather than other ER membrane proteins. What is missingin support of the conformational coupling idea in general isclear evidence showing a required colocalization of storesand sites of Ca2+ influx.

3. Ion selectivity and conductance: a useful yardstickfor judging candidate CRAC channel genes

Several genes have been proposed in recent years to en-code the CRAC channel, based on imaging or patch-clampstudies of cells induced to overexpress candidate genes. Athorough characterization of the conductive pores of thesechannels can provide the kind of definitive evidence thatis needed to test them as candidates. Fluorescence imag-ing measurements, in which the only readout is the rate ofion binding by intracellular reporter dyes, as well as electri-cal measurements of total membrane conductance, are not


generally able to distinguish among related channels withsubtly different subunit compositions or properties. Recentstudies of ion permeation through CRAC channels have shednew light on the basic properties of its pore. These results,discussed below, have important implications for the identityof CRAC channel genes, the nature of the Ca2+ signals theygenerate, and the molecular mechanisms governing their ionselectivity.

3.1. CRAC is a highly Ca2+ selective, low conductancechannel

The CRAC channel selects for Ca2+ over monovalentcations by a ratio of∼1000:1, as judged by the proportionof the current carried by Ca2+ under physiological ionicconditions [56,57]. This degree of selectivity is matchedonly by the voltage-gated Ca2+ channel family. UnlikeCaV channels, however, CRAC channels have an extremely

Fig. 2. Estimating the size of unitary Na+ and Ca2+ currents through CRAC channels using fluctuation analysis.ICRAC was induced by treatment with1�M TG and was recorded at a constant holding potential of−110 mV. (A) Mean value and variance of CRAC current in response to 2-APB (5�M) andremoval of divalent cations. Each point represents the mean or variance calculated from 200 ms current traces. (B) Sample 200 ms trace of Na+-ICRAC

as it depotentiates in the presence of DVF Ringer’s. Note the absence of obvious single-channel openings or closings. The zero-current level is indicatedby the dashed line. (C) Mean-variance analysis of Na+-ICRAC. The plot shows the mean value and variance of 200 ms current sweeps collected asNa+-ICRAC depotentiated in DVF Ringer’s from the experiment in A. The data are fit by a line with a slope of−31.2 fA. (D) Mean-variance analysisof Ca+-ICRAC. Mean and variance values were calculated as Ca+-ICRAC was enhanced by 2-APB in 20 mM Ca2+ Ringer’s from A. The data are fit bya line with a slope of−3.9 fA. Adapted from[58].

small unitary conductance for Ca2+. The conductanceappears to be too small to enable single-channel currentmeasurements, and has been estimated to be on the or-der of 10–20 fS based on current noise analysis[10,58](Fig. 2).

This extremely high Ca2+ selectivity and extremely smallconductance, conspicuous hallmarks of the CRAC channel,have important implications for their function in the T cell.First, the lack of significant Na+ current through CRAC un-der physiological conditions makes it a particularly efficientCa2+ influx pathway. Given that [Ca2+]i elevations lastingup to hours are required to trigger T-cell activation, minimiz-ing the influx of Na+ over such extended periods may benefitthe cell by lessening the energetic drain of having to pumpNa+ back out. Second, the small conductance of CRACchannels may influence the specificity of Ca2+ signaling inthe cell. In excitable cells, there are numerous examples oflocal signaling by Ca2+ that occurs through the close spatial


coupling of CaV channels and targets such as KCa chan-nels, ryanodine receptors, calmodulin, and synaptic vesicles[59,60]. In T cells, Ca2+ entering through CRAC channelsacts locally to trigger fast inactivation ofICRAC [44] andmodulate the activity of plasma membrane Ca2+-ATPases[61] (Bautista and Lewis, in preparation). The extremelysmall conductance of CRAC channels may help to explainwhy single CRAC channels inactivate independently[44],unlike the more conductive CaV channels which engage incrosstalk[62]. Thus, local signaling by CRAC channels maybe much more spatially restricted and tightly controlled thanlocal signaling by voltage-gated Ca2+ channels.

3.2. Ca2+-dependent potentiation of ICRAC: a vexingproblem for ion permeation studies

CRAC channels require extracellular Ca2+ in order tomaintain maximal activity in response to store depletion.This behavior, originally described as Ca2+-dependentpotentiation, or CDP, is manifest as a time-dependent,several-fold increase inICRAC amplitude occurring within10–20 s of Ca2+ readdition to cells with depleted stores[63,64]. Removal of extracellular Ca2+, or replacementwith divalent ions that do not support potentiation well (e.g.Ba2+ or Sr2+), causes a slow decline in channel activity.The mechanism of CDP is not understood. Its extent in-creases with membrane hyperpolarization, suggesting thatthe underlying Ca2+ binding site may reside within thepore of the channel. The site is not likely to be intracel-lular, as CDP is unaffected by intracellular BAPTA, andextracellular Ni2+ can substitute for Ca2+ in potentiatingCRAC channels even though it is not thought to permeatethe channels appreciably[64].

CDP has presented a unique set of problems for stud-ies of ion permeation in CRAC channels, because it causeschannel activity to drop following substitution of Ca2+ byother ions. Thus, steady-state current measurements tend togreatly underestimate the conductance of CRAC channelsfor divalent ions that do not support potentiation. For exam-ple, ICRAC declines over time after Ba2+ is substituted forCa2+; thus, measurements of the steady-state current haveled to the widespread belief that CRAC channels conductBa2+ poorly, however, measurements of peak Ba2+ currentshow that it actually conducts Ba2+ even better than Ca2+[63] (Prakriya and Lewis, in preparation). CDP has also com-plicated attempts to record single-channel CRAC currentscarried by Na+ under divalent-free conditions, as describedbelow.

3.3. Mg2+-inhibited cation (MIC) channels and areevaluation of CRAC pore properties

The ability to measure currents through single CRACchannels would be a powerful tool in understanding the reg-ulation of CRAC channels at a molecular level. Access tothe intracellular side of the channel in excised patches would

enable direct application of agents to probe channel regula-tion, and detailed information about effects on channel gat-ing could be obtained from kinetic single-channel measure-ments. The small conductance of CRAC channels to Ca2+precludes single-channel measurements, but it is known thatthe current through highly selective CaV channels can be en-hanced by an order of magnitude by removing extracellulardivalent ions and allowing Na+ to permeate[65,66]. CRACchannels behave in a similar way[56,58,67,68], but becausethe current slowly declines in the absence of Ca2+ by ∼90%(due to depotentiation as discussed above), the steady-stateNa+ current is not significantly larger than the Ca2+ cur-rent. This conundrum appeared to be overcome by the find-ing that removal of intracellular Mg2+ increases the size andlongevity of the Na+ current, making it∼25 times largerthan the CRAC Ca2+ current[69]. Under these conditions,the whole-cell current slowly activates due to the progres-sive, all-or-none opening of single 40-pS channels to a highopen probability of >0.9[70]. The 40-pS Na+-conductingchannels were considered to be CRAC channels based ontheir slow time course of activation and inhibition by extra-cellular Ca2+, Mg2+, Ni2+, and Gd3+ [41,70,71]. Overall,this discovery presented exciting new opportunities for themolecular characterization of CRAC channels and their acti-vation mechanism. Indeed, the conductance, selectivity, andhigh open probability of the Na+-conducting channels weresoon used as a foundation for studying CRAC channel acti-vation in patches and cells[41,72], quantifying changes inCRAC channel expression during T-cell activation[71], andtesting a candidate gene for the CRAC channel pore[73].

Subsequent studies by several groups have establishedthat the 40-pS channels are not in fact CRAC channels, butinstead are a distinct cation channel activated by the removalof intracellular Mg2+ [58,74,75]. This current (IMIC) differsfrom ICRAC in several key respects, summarized inTable 1. Itis not store-dependent, it is blocked by spermine, it is muchless sensitive than CRAC to inhibition by SKF 96365 and2-APB, and it is inhibited by intracellular Mg2+, MgATP,or GTP�S. A number of differences in ion selectivity werealso found, including a much greater permeability to Cs+relative to CRAC channels. The large number of properties(activation mechanism, pharmacology, and ion selectivity)that distinguish the current fromICRAC make it unlikely thatremoval of intracellular Mg2+ merely alters CRAC channelproperties. Rather, it is more likely that the current is dueto the activity of a distinct type of channel. This conclusionis strongly supported by genetic evidence, as Jurkat cellmutants and human SCID T cells lackICRAC but expressnormal levels ofIMIC [58] (Prakriya and Lewis, unpublishedobservations).

The activity of the 40-pS channels can be inhibitedin excised patches by Mg2+ on the intracellular side,and in whole-cell recordings by inclusion of 8–10 mMMg2+. Based on their cation sensitivity and sensitivity toMg2+, we refer to the channels as Mg2+-inhibited cation(MIC) channels. The same channels are also inhibited


Table 1Distinguishing properties of CRAC and MIC channels

Property CRAC channels Reference MIC channels Reference

Store-dependent activation Yes See text No [58,74]Inhibition by Mgi

2+ or MgATP No [58] Yes [58,74,75]Inhibition by GTP�S Only if applied before store depletion [11] Yes [74]Rectification (with Cao2+ + Mgo

2+) Inward [58,74,75] Mostly Outward [58,74,75]Conductance to divalents Ca2+, not Mg2+ [5,10,56] Ca2+ + Mg2+ [76]Selectivity (PCs/PNa) 0.13 [58,68] 1.2 [58]Duration in DVF conditions Transient [58] ∼Sustained (with 0 Mgi 2+) [58,74]Inhibition by SKF 96365 (20�M for 120 s) 87± 4% [58] 10 ± 2% [58]Inhibition by 2-APB (IC50) ∼10�M, irreversible [42,74] ∼50�M, quickly reversible [58,74]Enhancement by 2-APB (EC50) ∼3�M [42] None [58]Inhibition by spermine No [75] Yes [75]Unitary current (Na+, −110 mV) −31 ± 2 fA [58] −4.5 ± 0.4 pA [58]Unitary chord conductance (Na+, −110 mV) ∼0.2 pS [58] 44 ± 3 pS [58]

by exogenous MgATP introduced via the patch pipette,leading to the early suggestion that they are regulatedby Mg2+ + cellular ATP and prompting the name Mag-NuM, for Mg2+ nucleotide-regulated Metal channel[76].Based on ion selectivity and single-channel conductance,MIC/MagNuM channels are most likely encoded by thetrpm7 gene, which is an essential gene widely expressed incells, including Jurkat and RBL cells[76,77]. Although itcontains an intrinsic kinase domain in its cytoplasmic C ter-minus, the regulation of this channel in cells by MgATP isnot well established[76,77]. We have found that depletionof cellular ATP fails to activate MIC current in Jurkat Tcells, raising the possibility of complex regulation (Prakriya,Bautista, and Lewis, in preparation). Complex regulation isalso suggested by the slow time course of current inductionfollowing rapid removal of Mgi2+ [58], acceleration ofIMICrundown by SKF96365[75], and potent effects of PIP2 de-pletion on TRPM7 current[78]. Clearly, additional studiesare required to elucidate the mechanisms of regulation andfunctional contributions of this interesting channel.

CRAC channel properties (CDP, ion selectivity and recti-fication) are not affected significantly by intracellular Mg2+or MgATP [58,68,74,75]. Thus, inhibition ofIMIC by thesecompounds was used to study the Na+ current throughsingle CRAC channels. Interestingly, the Na+ conductanceof single CRAC channels is too small to be resolvable inwhole-cell recordings, indicating that the 40-pS monovalentconductance previously attributed to the CRAC channel wasincorrect. Noise analysis of whole-cell Na+-ICRAC indicateda conductance of∼0.2 pS[58], a figure that is∼10-foldlarger than the conductance for Ca2+.

These results forced a significant revision in the bio-physical fingerprint of the CRAC channel, with importantimplications for the interpretation of previous studies of theregulation and molecular identity of CRAC channels. Forexample, the upregulation of CRAC channel expression dur-ing T-cell activation[71], the voltage-dependent block ofmonovalent CRAC currents by extracellular Ca2+ and Mg2+[69,71], and inhibition of single CRAC channels by cyto-

plasmic Mg2+ and Ca2+ [41] were all measured under con-ditions that optimized activation ofIMIC. Thus, those resultsoffer information about MIC channels rather than CRAC.The small conductance of CRAC channels also distinguishesit from the “CRAC-like” currentImin, which is thought tobe activated through conformational coupling and has aunitary conductance of 1 and 6 pS for Ca2+ and Na+, re-spectively[16]. Finally, based on the ratio of whole-cell andsingle-channel Na+ current through the CRAC channel, weestimate that Jurkat T cells express >5000 channels per cell[58], which is similar to a previous estimate based on Ca2+currents [10], but substantially higher than the 100–400channel estimate based on the 40-pS MIC channel[70]. Thenew data on CRAC channel pore properties also have impli-cations for the mechanism of ion selectivity as well as fortesting putative CRAC channel genes, as described below.

3.4. Mechanisms of ion selectivity and permeationfor CRAC channels

Much evidence suggests that CRAC channels and CaVchannels use a similar mechanism for achieving extremelyhigh selectivity for Ca2+ under physiological ionic condi-tions. As was first shown for CaV channels[65,66], thecomplete removal of external divalent ions renders CRACchannels freely permeable to Na+ [56,58,67,68]. BecauseNa+ and Ca2+ have similar atomic diameters (∼2 Å) thisbehavior implies that Ca2+ selectivity derives from some-thing other than a sieve-type mechanism based on ion sizealone. Instead, the CRAC channels appear to achieve highCa2+ selectivity by high affinity binding of Ca2+ within thepore of the channel, which prevents the passage of Na+ ions[67,68]. As extracellular [Ca2+] is increased from zero intothe micromolar range, current through CRAC channels de-creases as Ca2+ blocks flow of Na+ ions, and then as [Ca2+]ireaches millimolar levels the current increases as Ca2+ be-gins to flow through the channel. This “anomalous molefraction” behavior is a common hallmark of multi-ion pores[79]. Thus, in physiological solutions, CRAC channels (like


CaV channels) select for Ca2+ over the more prevalent Na+via high-affinity binding of Ca2+ to the selectivity filter.

For CaV channels, the permeability sequence based onbiionic reversal potentials is Ca2+ > Sr2+ > Ba2+, whilethe conductance sequence is Ba2+ > Sr2+ > Ca2+, basedon current amplitudes at negative potentials. These resultshave been interpreted to mean that Ca2+ is more permeantbut binds more tightly than the other divalent ions to theselectivity site, leading to a lower throughput rate[79]. Inpreliminary experiments using a rapid solution exchange tomeasure peak Ba2+ and Sr2+ currents, we have found thatCRAC channels display the same behavior (Prakriya andLewis, in preparation). These data suggest the possibility ofstructural similarities between the selectivity filters of thetwo channels.

Given these close similarities, it is surprising that in com-parison to CaV channels, the conductance of CRAC chan-nels for both Ca2+ and Na+ is ∼500-fold smaller, and Cs+permeability of CRAC is∼five-fold smaller (PCs/PNa ∼0.1). One clue to explain this difference may be providedby estimates of pore diameter. By measuring the perme-ability of NH4

+ and its methylated derivatives, the diam-eter of CaV channels has been estimated to be∼6 Å [80].Using a similar approach, we have estimated the diameterof the narrowest part of the CRAC channel pore in Jurkatcells to be only∼3.8 Å (Prakriya and Lewis, in prepara-tion), and an estimate of 3.2–5.5 Å was reported in RBLcells [68]. Whether this figure reflects the diameter of theselectivity filter itself or a different part of the pore remainsto be investigated. Given that the atomic diameter of Cs+is ∼3.4 Å, the small pore diameter might explain why Cs+is practically impermeant through CRAC channels. In addi-tion, the small pore diameter may help to explain the lowconductance of CRAC channels for Ca2+ and Na+, if itpresents a relatively non-selective rate-limiting barrier act-ing in series with the selectivity filter. Structure–functionstudies using altered CRAC channel genes will be essentialfor testing these ideas and achieving a deeper understand-ing of the mechanism of ion selectivity and permeation byCRAC channels.

3.5. TRP homologs as potential CRAC channels

Interest in TRP proteins as store-operated Ca2+ channelsgrew out of early work onDrosophila TRP, which wasoriginally hypothesized to be store-dependent based on itsCa2+ permeability and the requirement for PLC� in TRPactivation by light. Evidence that mammalian homologs ofTRP are store-dependent channels has been contradictory,with explanations that include differences in cell type orlevel of expression. Somewhat ironically, abundant evidencehas accrued in the meantime showing that activation ofDrosophila TRP occurs via a store-independent mechanismprobably involving lipid mediators[81–83]. Most of theTRP homologs that have been proposed to encode SOCs(TRPC1, TRPC3, TRPC4), when expressed in heterologous

systems, do not display the same high Ca2+ selectivity ofthe CRAC channel, suggesting that homomultimers of theseproteins do not comprise the CRAC channel[38]. However,the current practice of characterizing singletrp genes ex-pressed heterologously in cells that have endogenous SOCsmay be inherently unable to address the question of whetherCRAC contains one or more TRPs. TRPs are known to formheteromultimers in vitro and in vivo[84–86], in some casesgenerating altered pore properties. Thus, even a true CRACsubunit when expressed heterologously may fail to generatea CRAC-like current, either by combining with the “wrong”endogenous subunits, or by failing to form the “right”associations.

A good recent example of this approach relates to TRPV6(CaT1, ECaC2), a member of the vanilloid TRP family.trpv6 is expressed in Jurkat T cells and RBL cells, and whenoverexpressed in CHO cells does appear to show many ofthe biophysical properties of the CRAC channel, includ-ing high selectivity for Ca2+, anomalous mole fraction be-havior, and fast inactivation. These similarities plus someevidence for store-dependent activation led to the sugges-tion that it comprises all or part of the CRAC channel pore[73]. Further work, however, shows that TRPV6 alone can-not account for a more complete set of CRAC permeationcharacteristics. Under divalent-free conditions, TRPV6 hasweaker inward rectification and a higher Cs+ permeability(PCs/PNa = 0.48 versus 0.12 for CRAC), and is blockedin a voltage-dependent manner by intracellular Mg2+ [87].Perhaps the most obvious discrepancy is the single-channelconductance: under divalent-free conditions, TRPV6 has aNa+ conductance of 42–58 pS[73], >100-fold larger thanthe CRAC channel (∼0.2 pS). Thus, TRPV6 alone cannotaccount for the properties of the CRAC channel conductionpathway. But could it participate with another subunit? Sucha scenario would help to explain a recent report that over-expression of a non-conducting pore mutant of TRPV6 canpartially suppress endogenousICRAC in transfected Jurkatcells [88]. These promising results suggest that TRPV6 cancombine with endogenous CRAC channel subunits, but fur-ther studies will be needed to rule out non-specific effectsof these constructs, and to show directly whether TRPV6normally participates in forming the channel.

Another recent report has shown that the genetic deletionof TRPC1 in DT40 cells attenuates store-operated Ca2+ in-flux [89]. Interestingly, patch-clamp recordings indicate thatICRAC is severely reduced, but only in 80% of the cells;the remaining 20% express CRAC currents indistinguish-able from wild type. A proposed explanation is that knock-out of TRPC1 induced compensatory changes in expressionof a redundant subunit, which reached an all-or-none thresh-old for fully rescuing the channels in 20% of the cells[89].The existence of such an all-or-none process in CRAC chan-nel activation is not well established (see above), but fur-ther characterization of the properties of the current in thesemutant cells may uncover subtle alterations consistent withsubstitution of a redundant subunit.


4. Future directions

Thus far, the search for a molecular mechanism for theCRAC channel has consisted primarily of making a guessas to what molecules or genes might be involved, and thenthrough overexpression, knockdown, or pharmacological in-tervention trying to amass evidence in support of this guess.Heterologous expression studies focused on the TRP familythus far have failed to reveal a gene that can recreate all of theproperties of the CRAC channel or even its pore. However,it may be premature to rule out a role for several of thesegiven that CRAC may be a heteromultimer, and that manyof the expression systems possess endogenous versions ofSOCs that may recombine with transfected subunits. Phar-macological approaches to understand how store depletiontriggers channel activation have been fraught with problemsof interpretation, because many of the compounds that af-fect ICRAC have multiple targets, and it is often difficult todistinguish between effects on the channel from those on theactivation mechanism.

Less biased approaches in the search for genes encod-ing the CRAC channel and its activation machinery exist.A biochemical approach based on isolating the compo-nent proteins is currently hampered by the lack of specific,high-affinity ligands or a particularly rich source of CRACprotein. However, genetic approaches may be particularlyhelpful, considering that loss-of-function mutations in theCRAC pathway are not lethal to cells[8,23,24]. Thus, futureattempts to complement CRAC-deficient mutant cells or toidentify and mutate store-operated CRAC-like conductancesin genetically tractable organisms likeDrosophila and C.elegans may offer a fresh perspective on the molecular basisof store-operated Ca2+ entry.

Acknowledgements

The work from the authors’ lab cited in this article wassupported by a postdoctoral fellowship from the IrvingtonFoundation for Immunological Research (to M.P.) and NIHgrant GM45374 (to R.S.L.).

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CRAC channels: activation, permeation, and the search for a molecular identity