A role for Orai in TRPC-mediated Ca2� entry suggeststhat a TRPC:Orai complex may mediate store andreceptor operated Ca2� entryYanhong Liaoa,1, Nicholas W. Plummera, Margaret D. Georgea, Joel Abramowitza, Michael Xi Zhub,and Lutz Birnbaumera,2

aLaboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health,Department of Health and Human Services, Research Triangle Park, NC 27709; and bDepartment of Neuroscience and Biochemistryand Center for Molecular Neurobiology, Ohio State University, Columbus, OH 43210

Contributed by Lutz Birnbaumer, December 31, 2008 (sent for review December 25, 2008)

TRPC and Orai proteins have both been proposed to form Ca2�-selective, store-operated calcium entry (SOCE) channels that areactivated by store-depletion with Ca2� chelators or calcium pumpinhibitors. In contrast, only TRPC proteins have been proposed toform nonselective receptor-operated calcium entry (ROCE) cationchannels that are activated by Gq/Gi-PLC� signaling, which is thephysiological stimulus for store depletion. We reported previouslythat a dominant negative Orai1 mutant, R91W, inhibits Ca2� entrythrough both SOCE and ROCE channels, implicating Orai partici-pation in both channel complexes. However, the argument for Oraiparticipating in ROCE independently of store depletion is tenuousbecause store depletion is an integral component of the ROCEresponse, which includes formation of IP3, a store-depleting agent.Here we show that the R91W mutant also blocks diacylglycerol(DAG)-activated Ca2� entry into cells that stably, or transiently,express DAG-responsive TRPC proteins. This strongly suggests thatOrai and TRPC proteins form complexes that participate in Ca2�

entry with or without activation of store depletion. To integratethese results with recent data linking SOCE with recruitment ofOrai and TRPCs to lipid rafts by STIM, we develop the hypothesisthat Orai:TRPC complexes recruited to lipid rafts mediate SOCE,whereas the same complexes mediate ROCE when they are outsideof lipid rafts. It remains to be determined whether the moleculesforming the permeation pathway are the same when Orai:TRPCcomplexes mediate ROCE or SOCE.

diacylglycerol � STIM1 � store operated calcium entry �transient receptor potential

The role of Orai proteins in store operated Ca2� entry hasbeen firmly established (for reviews see refs. 1–3). Recent

studies suggest that Orai forms a tetrametic store operated Ca2�

channel (4, 5) and this formation may be aided by STIM1, asensor for endoplasmic reticulum Ca2� content, which facilitatesthe dimerization of preexisting Orai dimers in the plasmamembrane (5). However, there also exists a body of evidencestemming from transient (6) and stable (7) overexpressionstudies, as well as acute (8), transient (9), and stable (10, 11)suppression studies that strongly implicate participation ofTRPC channels in store operated Ca2� entry.

Store operated Ca2� entry (SOCE) is closely related toreceptor operated Ca2� entry (ROCE), and it is not known towhat extent store operated Ca2� entry contributes to entry ofCa2� after activation of phospholipase C (PLC) in response toexposure of cells or tissues to agents that trigger phosphoinosi-tide hydrolysis with formation of inositol 1,4,5-trisphosphate(IP3) plus diacylglycerol (DAG). From the electrical viewpoint,activation of receptors coupled to intracellular events by the Gqor Gi class of G proteins is followed by activation of a nonse-lective cation current (cf. 12, 13). This current is transient andinactivates with a time course resembling that reported foractivation of a Ca2�-release activated Ca2� current (Icrac)

(13–15). Ca2� entry during this time increases and continues foras long as agonist is present (16), consistent with a switch fromentry carried by nonselective Ca2� permeable cation channelsformed by TRPCs to entry mediated by CRAC channelsproposed to be formed by TRPCs or Orai proteins, or, as wehave proposed, formed by complexes of TRPCs with Oraiproteins (17).

Mammalian Orai1 was discovered by 2 approaches: an RNAisuppression screen (15) and the combination of an RNAisuppression screen and positional cloning of a familial mutationresponsible for severe combined immunodefficiency (SCID; ref.18). Not surprisingly, expression of the SCID mutant in normalHEK-293 cells interferes with thapsigargin-stimulated SOCE(19), presumably by interfering with the formation of normalOrai1 multimers. The participation of TRPCs in store operatedCa2� entry involving Orai1 was inferred by us from studies inwhich expression of exogenous Orai1 increased thapsigargin-stimulated Ca2� entry only in cells stably overexpressing a TRPC(TRPC3 or TRPC6) but not in control cells (17, 19), i.e., byshowing an effect of Orai1 that is dependent on the TRPC statusof the cells.

Here we expand on these studies by providing further con-nections between Orai1 and TRPCs. We took advantage of thefact that a low concentration of GdCl3 (1 �M) blocks SOCE, butnot ROCE mediated by TRPC channels, to show that Orai1,expressed at the level that enhances SOCE, leads to appearanceof Gd3�-resistant ROCE and that the SCID mutant of Orai1,Orai1[R91W], not only inhibits SOCE and ROCE in HEK293cells but also Ca2� entry elicited by activation of TRPC3 with1-oleoyl-2-acetyl-sn-glycerol (OAG). OAG-induced Ca2� entryis specific for the TRPC3 subfamily of TRPCs (TRPC3, TRPC6and TRPC7) (13, 20). We propose a model that incorporates ourdata and recent data from the literature indicating that SOCEappears to occur through channels located in lipid rafts whereasROCE may occur outside of the lipid rafts.

ResultsIn previous studies we had found that expression of low levels ofOrai1 [corresponding to a transfection with 50–60 ng of inputexpression plasmid in our transfection protocol (17)] enhancesSOCE in HEK-293 cells expressing TRPC3 or TRPC6 in stableform. We expanded our studies to 2 additional TRPCs,TRPC1and TRPC7, by testing the effect of Orai1 in cells stablyexpressing them. We demonstrate that Orai1 enhanced SOCE in

Author contributions: Y.L., N.W.P., M.D.G., J.A., and L.B. designed research; Y.L., N.W.P.,and M.D.G. performed research; M.X.Z. contributed new reagents/analytic tools; Y.L.,N.W.P., M.D.G., J.A., and L.B. analyzed data; and J.A., M.X.Z., and L.B. wrote the paper.

The authors declare no conflict of interest.

1Present address: Department of Anatomy, Tongji Medical College, Huazhong University ofScience and Technology, Wuhan 430030, China.

2To whom correspondence should be addressed. E-mail: birnbau1@niehs.nih.gov.

3202–3206 � PNAS � March 3, 2009 � vol. 106 � no. 9 www.pnas.org�cgi�doi�10.1073�pnas.0813346106

Dow

nloa

ded

by g

uest

on

Mar

ch 1

, 202

1

both cell lines, with the exception that cells expressing TRPC7responded to 5-fold lower inputs of Orai1. As a result, at plasmidlevels that are optimal for TRPC3 expressing cells, the TRPC7cells were either unaffected or slightly inhibited by Orai1 (Figs.1 and 2).

We reported further that mutant Orai1 not only inhibitedSOCE but also ROCE (19). These findings strongly suggestedthat the physiological roles of Orai may not be restricted to itsparticipation in SOCE. However, because ROCE includes anaspect of SOCE triggered by IP3-induced store depletion, wesought another method to stimulate TRPC function that wouldnot include a store depletion phase. One such form of Ca2� entrymediated by TRPC is the DAG-stimulated Ca2� entry seen incell lines expressing one of the members of the TRPC3 subfamilyof TRPCs. We found that the SCID mutant inhibits the OAG-activated Ca2� entry. This is shown for TRPC3 expressed stably(Fig. 3A) or transiently (Fig. 3B) and was seen also in TRPC6expressing cells (data not shown). Wild-type Orai1 did not affectCa2� entry stimulated by OAG (Fig. 3A).

Finally, we reported also that expression of high levels of Orai1and STIM1 cause the appearance of Gd3�-resistant ROCE (19).This phenomenon was explored further. We found that, eventhough our initial experiments indicated that both STIM1 andOrai1 were needed at high concentrations such as those achievedwith 1 �g of each of the input expression plasmids, Orai1 canelicit Gd3�-resistant ROCE at low levels of expression in theabsence of STIM1 (Fig. 4 Right) and that Gd3�-resistant ROCE

can be generated in HEK293 cells by transfecting as little as25–50 ng of the Orai1 expression plasmid (Fig. 5). This is thesame amount as was used to obtain TRPC-dependent enhance-ments of SOCE in cells expressing exogenous TRPCs in stableform. High amounts of the Orai1 expressing plasmid are inhib-itory even in the presence of Gd3� (bottom trace of Fig. 5).

In other experiments we found by real-time qPCR thatcoexpression of high levels of Orai1 and STIM1, which enhancedSOCE by more that 10-fold (19), did not significantly affect theendogenous levels of mRNAs coding for expression of endog-enous TRPC1, TRPC3, TRPC4, or TRPC6 mRNA, which arethe TRPC channels expressed in our HEK-293 cells (data notshown).

DiscussionFig. 6 summarizes our hypothesis that the location of a TRP-C:Orai complex determines whether it functions as a SOCE ora ROCE channel. TRPC:Orai channels activated outside theconfines of lipid rafts are assumed to mediate ROCE, whereaschannels located in lipid rafts mediate SOCE. The conversionfrom ROCE to SOCE and translocation from the nonraft to theraft domains may be mediated through STIM binding and

Fig. 1. Enhancement of SOCE in HEK293 cells stably expressing TRPC1 (T1–8cells). Cells were cotransfected in 60 mm dishes with empty pcDNA3 (Clontech)or 60 ng of pOrai1 (pcDNA3 carrying the cDNA of wild-type Orai1 under thecontrol of the CMV promoter) and peYFP as described (17). After 24 h, cellswere replated onto coverslips for an additional 24 h. SOCE was monitored incells loaded with Fura2 by dual wavelength ratiometric fluorescence videomicroscopy as described (44, 17).

Fig. 2. Effect of varying the input pOrai1 on SOCE in HEK293 cells stablyexpressing TRPC7 (T7–2 cells).

A B

Fig. 3. (A) Orai1[R91W] inhibits Ca2� entry triggered by OAG (100 �g/ml) inHEK cells that stably express TRPC3 (clone T3H1), and (B) in HEK-293 cells thattransiently express TRPC3. (A) T3H1 cells were transfected with 0.1 �g peYFP(Control, deep blue), 0.1 �g peYFP plus 60 ng of pOrai1[R91W] (R91W,bluish-green), or with 0.1 �g peYFP plus 60 ng pOrai1 (wild-type Orai1, red)and analyzed for their response to OAG. (B) HEK-293 cells were cotransfectedwith 1.0 �g pTRPC3 and 0.1 �g peYFP (TRPC3, deep blue) or with 1.0 �gpTRPC3, 60 ng pOrai1[R91W] and 0.1 �g peYFP (TRPC3 � W91R, blue-green)and analyzed for their response to OAG. Transfections were in 60 mm dishesas described under Methods. The OAG responses were tested 48 h aftertransfection. The OAG activation protocol was as described (17). Similar resultswere obtained with HEK-293 cells stably expressing TRPC6 (data not shown).

Fig. 4. Wild-type (wt) Orai1 causes the appearance of Gd3�-resistant ROCEin HEK-293 cells and Orai1[R91W] inhibits ROCE both in the absence (Left) andthe presence (Right) of 1 �M Gd3�. Note: no exogenous TRPC was present.

Liao et al. PNAS � March 3, 2009 � vol. 106 � no. 9 � 3203

CELL

BIO

LOG

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 1

, 202

1

dimerization of the preexisting Orai dimers, which might be thepredominant form of TRPC:Orai channels that mediate ROCE.

Evidence has accumulated to indicate that the channel com-plexes responsible for SOCE and ROCE do not assemble in thesame membrane compartments. Specifically, store depletioncauses (i) STIM1 to aggregate into submembranous puncta(21, 22) and (ii) the translocation of Orai1 (23, 24) and TRPCs(25, 26) to these puncta. As a consequence, STIM1 and Orai1,as well as STIM1 and the TRPC molecules, come into closeenough proximity to appear as colocalized when viewed througha confocal microscope to allow for Foerster resonance energytransfer (FRET) from STIM1-CFP to Orai1-YFP (23, 27) andfrom STIM1-CFP to TRPC1-YFP (26) and to allow for 3-waycoimmunoprecipitation from normal human platelet lysates ofSTIM1 with Orai1 and TRPC1, of Orai1 with STIM1 andTRPC1, and of TRPC1 with STIM1 and Orai1 (28) or simplecoimmunoprecipitation of TRPC1 with STIM1 and Orai1 asseen in human submaxillary gland cells (29).

The channels assembled in this manner are not only aggregatesof the required proteins but concentrate in domains rich in cho-lesterol and sphingomyelins referred to as lipid rafts (24, 26, 30).Participation of a TRPC in thapsigargin-activated, store-operatedcurrents (SOC) had also been inferred from TRPC1 knockdownexperiments in human submaxillary gland cells (29) and fromTRPC1 knockout experiments in mouse salivary glands (11) inwhich SOCE was reduced.

Initial agonist induced ROCE, which by definition does notinvolve STIM1, finds TRPCs (26, 30) and Orai proteins (24)diffusely distributed in the nonlipid raft domains of the plasma

membrane from where they are recruited to lipid rafts byactivated STIM1. The slow development of Icrac is consistentwith the need for redistribution of plasma membrane proteinsunder the influence of activated STIM1 and assembly of theproteins forming the influx channel.

At the molecular level, the C terminus of STIM1 has beenshown to interact with the C terminus of Orai1 through theirrespective coiled coil domains (27) and with the C terminus ofTRPC1 through the interaction of the C-terminal KK dipeptideof STIM1 and a DD dipeptide of TRPC1 located very close tothe TRPC TRP box (31).

Assembly of the Orai:TRPC complex in lipid rafts appears tobe an obligatory event for the development of a store operatedcurrent SOC, as disruption of lipid rafts prevents the develop-ment of both the STIM1-to-TRPC1 FRET signal and the storedepletion-activated current (26).

Activation of ROCE is rapid with time lags in the order of afew seconds at most. It presumably arises from entry occurringoutside of lipid rafts, although there is no a priori reason thatactivation could not also occur in the environment of lipid rafts.We presume that ROCE is, at least initially, carried by TRPCchannels activated secondarily to PLC activation. Despite inten-sive studies, a unifying mechanism by which PLC activation leadsto TRPC activation has not yet been found. Some TRPCs can beactivated by DAG formed by the Gq/Gi-activated PLC� [theseinclude TRPC2 (32), TRPC3 (13), TRPC6 (13), TRPC7 (20) andpossibly also Drosophila TRP channels (33)]. Other TRPCs, e.g.,TRPC4, TRPC5, and TRPC1, do not appear to be activated bya DAG. All TRPCs have the potential to interact with anN-terminal region of the IP3 receptor (34, 35), which, as shownfor TRPC3, has the ability to displace inhibitory Ca2�/calmod-ulin (36). Yet, this is still not likely to be a common mechanismof activation of all TRPCs, including Drosophila TRPs, asDrosophila photoreceptor cells lacking the IP3 receptor areactivated by light (37). We hypothesize that rapid activation ofTRPC channels may arise therefore by a combination of DAG-mediated activation and a protein::protein interaction between theTRP channel molecules and PLC�. In support of the latter, TRPC4has been coimmunoprecipitated with PLC� from mouse brainlysates (38), and TRPC1 and PLC� have been coimmunoprecipi-tated with caveolar complexes from salivary gland cell lysates (39).

Fig. 6. Model of TRPC (purple):Orai (red) complexes operating as ROCEchannels outside of lipid rafts and as SOCE (CRAC) channels within the confinesof lipid rafts to which the complex is recruited by the multifunctional Cterminus of activated STIM1 (blue; ref. 26). STIM1 is shown interacting bycoiled-coiled domain interaction with the C terminus of Orai1 (yellow; ref. 27)and by ionic interaction of its C-terminal KK dipetide with the DD dipeptide ofTRPC (light blue; ref. 31). The IP3 receptor (cyan) is shown in its IP3-activatedform that confers to it the ability to interact with a region of TRPCs located intheir C-termini (pink; ref. 34).

Fig. 5. Induction of Gd3�-resistant ROCE by varying the amount of inputpOrai1 shown in ng per 60 mm dish. All transfections also contained constantamounts of plasmids directing the expression of exogenous V1a vasopressinreceptor and peYFP. At the concentrations tested the Gd-resistant ROCE wasmaximal at 50 ng pOrai1 (trace #5). Ten-fold higher amounts of plasmid wereinhibitory (trace #6). Trace #1, (ROCE in the absence of Gd3�) was obtainedfrom cells transfected only with peYFP and pV1aR. Note: that no exogenousTRPC was present.

3204 � www.pnas.org�cgi�doi�10.1073�pnas.0813346106 Liao et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

, 202

1

One of our current, rather unexpected findings is the inter-ference by Orai1[R91W] with activation of TRPC3 and TRPC6by OAG. This finding adds Orai1 to TRPCs in the picture ofROCE. One would not expect Orai1[R91W] to interfere in adominant negative way with OAG activation if a multimeric Oraiwere not part of the channel formed by TRPCs. We propose thatOrai:TRPC complexes operating in lipid rafts are inhibited byGd3�, and those operating outside of lipid rafts, if formed byOrai1:TRPC3/6/7, show resistance to the lanthanide.

Neither expression of low levels of Orai1 nor that of STIM1in HEK293 cells (19) affect their SOCE. Expression of highlevels of Orai inhibits SOCE. Coexpression of high levels ofOrai1 and STIM1 relieves the inhibition by excess Orai1 andincreases SOCE at least 10-fold (19, 40–43). Orai and STIM1levels are therefore balanced in HEK-293 cells. As a result,appearance of Gd3�-resistant channels upon expression of lowlevels of Orai1 requires association to preexisting ‘‘unpaired’’TRPC channels.

The other finding reported here is the appearance of Gd3�-resistant ROCE upon expression of Orai1. Coexpression ofOrai1 and STIM1 leads to greatly enhanced SOCE and Icrac.Thus, expression of Orai1 in a cell expressing superstoichiomet-ric levels of a TRPC from the TRPC3 subfamily could lead to theaccumulation of Gd3�-resistant TRPC:Orai1 complexes as wefound (Figs. 4 and 5), which we presume to occur outside of lipidrafts (Fig. 6). Recruitment to lipid rafts depends on STIM1 (26).Thus, under condition of ROCE, and without an overexpressionof STIM1, the assembly of the CRAC conformation of thecomplex is not favored.

Fig. 7 presents a pictorial view of the sequence of events thatwe propose lead from a resting cell to a stimulated cell, firstallowing Ca2�to enter through the ROCE configuration of theTRPC:Orai channel followed by reconfiguration into a CRACchannel within the confines of lipid rafts. Although the data isnot shown, the initial ROCE channel may be activated not onlyoutside of lipid rafts but also within their confines.

Further studies are required to describe more accurately themolecular makeup of the channels mediating ROCE, includinganswering the question as to which of the molecules carries theionic currents. In the most formal analysis, there is the sameevidence available for TRPCs forming ion channels as for Oraiproteins forming ion channels.

MethodsCell and Molecular Biology. For transfections, cDNAs coding for eYFP (Clon-tech), V1a vasopressin receptor (V1aR), myc-tagged Orai and mutants, andSTIM1 were placed into pcDNA3 (Invitrogen). For expression of proteins inHEK and HEK derived cells, cells were grown in 60 mm dishes to ca. 80%confluence and were transfected (Lipofectamine method, Invitrogen) with amixture of plasmids that included plasmids coding for eYFP (0.1–0.5 �g), theindicated plasmids coding for wild-type or mutant Orai1, TRPC3 tagged withthe HA epitope at the C terminus, or combinations thereof and varyingamounts of pcDNA3 without insert (empty vector) to give 5.0 �g total plasmidDNA. For ROCE measurements, the V1aR cDNA in pcDNA3 (pV1aR, 1.5 �g) was

included in the transfection mixture. For measurement of [Ca2�]i, the cellswere replated 24 h after the transfection onto polylysine coated coverslips andallowed to recover for another 24 h before preparing them for SOCE or ROCEmeasurements. Loading with Fura2 was done by incubation with 2 �g Fura2a.m. in Hepes buffered saline for 30 min at 37 °C. Changes in [Ca2�]i weremonitored by dual excitation fluorescence ratiometric video microscopy asdescribed in refs. 44 and 17.

Quantitative reverse transcription-PCR (qPCR) was performed on total RNAfrom control mock transfected HEK-293 cells and Orai1 plus STIM1 transfectedHEK-293 cells, isolated 48 h after transfection, with reagents and gene specificprimers (TRPC1, cat # PPH15081A; TRPC3, cat #PPH12808E; TRPC4, cat#PPH15312A; TRPC5, cat #PPH14971A; TRPC6, cat# PPH13135A; TRPC7,cat#PPH15892A; GAPDH, PPH00150E) purchased from SuperArray BioscienceCorporation following the vendor’s instructions. Thermal cycling was per-formed in a BioRad iCycler using the cycling protocol suggested by the supplierof the reagents.

Other materials and all other methods were as in ref. 17.

ACKNOWLEDGMENTS. This work was supported by the Intramural ResearchProgram of the NIH (Z01-ES-101684).

1. Hogan PG, Rao A (2007) Dissecting Icrac, a store-operated calcium current. TrendsBiochem Sci 32:235–245.

2. Vig M, Kinet JP (2007) The long and arduous road to CRAC. Cell Calcium 42:157–162.3. Feske S (2007) Calcium signalling in lymphocyte activation and disease. Nat Rev

Immunol 7:690–702.4. Mignen O, Thompson JL, Shuttleworth TJ (2008) Orai1 subunit stoichiometry of the

mammalian CRAC channel pore. J Physiol 586:419–425.5. Penna A, et al. (2008) The CRAC channel consists of a tetramer formed by Stim-induced

dimerization of Orai dimers. Nature 456:116–120.6. Vannier B, et al. (1999) Mouse trp2, the homologue of the human trpc2 Pseudogene,

encodes mTrp2, a store depletion-activated capactitative Ca2� entry channel. Proc NatlAcad Sci 96:2060–2064.

7. Philipp S, et al. (1998) A novel capacitative calcium entry channel expressed in excitablecells. EMBO J 17:4274–4282.

8. Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR, Florman HM (2001) TRP2 is essentialfor Ca2� entry into mouse sperm triggered by Egg ZP3. Nat Cell Biol 3:499–502, 2000.

9. Zagranichnaya TK, Wu X, Villereal ML (2005) Endogenous TRPC1, TRPC3, and TRPC7proteins combine to form native store-operated channels in HEK-293 cells. J Biol Chem280:29559–29569.

10. Freichel M, et al. (2001) Lack of an endothelial store-operated Ca2� current impairsagonist-dependent vasorelaxation in TRP4-/- mice. Nat Cell Biol 3:121–127.

11. Liu X, et al. (2007) Attenuation of store-operated Ca2� current impairs salivary glandfluid secretion in TRPC1(-/-) mice. Proc Natl Acad Sci USA 104:17542–17547.

12. Hurst RS, Zhu X, Boulay G, Birnbaumer L, Stefani E (1998) Ionic currents underlyinghTrp3 mediated agonist-dependent Ca2� influx in stably transfected HEK293 cells.FEBS Lett 422:333–338.

13. Hofmann T, et al. (1999) Direct activation of human TRPC6 and TRPC3 channels bydiacyglycerol. Nature 397:259–263.

14. Hoth M, Penner R (1992) Depletion of intracellular calcium stores activates a calciumcurrent in mast cells. Nature 355:353–356.

15. Vig M, et al. (2006) CRACM1 is a plasma membrane protein essential for store-operatedCa2� entry. Science 312:1220–1223.

Fig. 7. Model of the events triggered by phospholipase C� activated by aGq/Gi-coupled GPCR (receptor signaling shown by red arrow). Nonacti-vated TRPC channels (1) are depicted as TRPC:Orai complexes based on ourfinding that spontaneous activities of TRPC3 channels (17) and TRPC6channels (C. Erxleben and D.L. Armstrong, personal communication) arereduced by expression of low levels of Orai1. The receptor-activated TRPC(2) is shown in association with Orai based on the finding reported herethat a dominant negative variant of Orai (Orai1[R91W]) inhibits ROCE aswell as DAG activated Ca2� entry. Activated STIM1 (3) is shown as a dimerwith a dual TRPC and Orai interacting C terminus (dark green) to reflectmultimerizaton and activation of its TRPC and Orai interacting abilityinduced by loss of Ca2� caused by active store depletion (4) triggered by IP3formed by PLC activity or passive store depletion without activation of PLC(light blue arrows). The activated CRAC channel (5) is shown in a lipid raftto which it is directed by activated STIM1 molecules. (Modified from figure6 in ref. 19).

Liao et al. PNAS � March 3, 2009 � vol. 106 � no. 9 � 3205

CELL

BIO

LOG

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 1

, 202

1

16. Liao CF, Schilling WP, Birnbaumer M, and Birnbaumer L (1990) Cellular responses tostimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells. J BiolChem 265:11273–11284.

17. Liao YH, et al. (2007) Orai proteins interact with TRPC channels and confer respon-siveness to store depletion. Proc Natl Acad Sci USA 104:4682–4687.

18. Feske S, et al. (2006) A mutation in Orai1 causes immune deficiency by abrogating CRACchannel function. Nature 441:179–185.

19. Liao Y, et al. (2008) Functional interactions among Orai1, TRPCs, and STIM1 suggest aSTIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl AcadSci USA 105:2895–2900.

20. Okada T, et al. (1999) Molecular and functional characterization of a novel mousetransient receptor potential protein homologue, TRP7. Ca2� permeable cation channelthat is constitutively activated and enhanced by stimulation of G protein coupledreceptor. J Biol Chem 274:27359–27370.

21. Wu MM, Buchanan J, Luik RM, Lewis RS (2006) Ca2� store depletion causes STIM1 toaccumulate in ER regions closely associated with the plasma membrane. J Cell Biol174:803–813.

22. Smyth JT, Dehaven WI, Bird GS, Putney JW, Jr (2008) Ca2�-store-dependent and-independent reversal of Stim1 localization and function. J Cell Sci 121:762–772.

23. Luik RM, Wu MM, Buchanan J, Lewis RS (2006) The elementary unit of store-operatedCa2� entry: Local activation of CRAC channels by STIM1 at ER-plasma membranejunctions. J Cell Biol 174:815–825.

24. Lioudyno MI, et al. (2008) Orai1 and STIM1 move to the immunological synapse and areup-regulated during T cell activation. Proc Natl Acad Sci USA 105:2011–2016.

25. Yuan JP, Zeng W, Huang GN, Worley PF, Muallem S (2007) STIM1 heteromultimerizesTRPC channels to determine their function as store-operated channels. Nat Cell Biol9:636–645.

26. Sampieri A, Zepeda A, Saldana C, Salgado A, Vaca L (2008) STIM1 converts TRPC1 froma receptor-operated to a store-operated channel: Moving TRPC1 in and out of lipidrafts. Cell Calcium 44:479–491.

27. Muik M, et al. (2008) Dynamic coupling of the putative coiled-coil domain of ORAI1with STIM1 mediates ORAI1 channel activation. J Biol Chem 283:8014–8022.

28. Jardin I, Lopez JJ, Salido GM, Rosado JA (2008) Orai1 mediates the interaction betweenSTIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2�

channels. J Biol Chem 283:25296–25304.29. Ong HL, et al. (2007) Relocalization of STIM1 for activation of store-operated Ca2�

entry is determined by the depletion of subplasma membrane endoplasmic reticulumCa2� store. J Biol Chem 282:12176–12185.

30. Pani B, et al. (2008) Lipid rafts determine clustering of STIM1 in ER-plasma membranejunctions and regulation of SOCE. J Biol Chem 283:17333–17340.

31. Zeng W, et al. (2008) STIM1 gates TRPC channels, but not Orai1, by electrostaticinteraction. Mol Cell 32:439–448.

32. Lucas P, Ukhanov K, Leinders-Zufall T, Zufall F (2003) A diacylglycerol-gated cationchannel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: Mecha-nism of pheromone transduction. Neuron 40:551–561.

33. Estacion M, Sinkins WG, Schilling WP (2001) Regulation of Drosophila transient recep-tor potential-like (TRPL) channels by phospholipase C-dependent mechanisms.J Physiol 530:1–19.

34. Boulay G, et al. (1999) Modulation of Ca2� entry by polypeptides of the inositol1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evi-dence for roles of TRP and IP3R in store depletion-activated Ca2� entry. Proc Natl AcadSci 96:14955–14960.

35. Tang J, et al. (2001) Identification of common binding sites for calmodulin and IP3receptors on the carboxyl-termini of Trp channels. J Biol Chem 276:21303–21310.

36. Zhang Z, et al. (2001) Activation of TRP3 by IP3 receptor through displacement ofinhibitory calmodulin from a common binding site. Proc Natl Acad Sci USA 98:3168–3173.

37. Acharya JK, Jalink K, Hardy RW, Hartenstein V, Zuker CS (1997) InsP3 receptor isessential for growth and differentiation but not for vision in Drosophila. Neuron18:881–887.

38. Tang Y, et al. (2000) Association of mammalian trp4 and phospholipase C isozymes witha PDZ domain-containing protein, NHERF. J Biol Chem 275:37559–37564.

39. Lockwich T, Singh BB, Liu X, Ambudkar IS (2001) Stabilization of cortical actin inducesinternalization of transient receptor potential 3 (Trp3)-associated caveolar Ca2� sig-naling complex and loss of Ca2� influx without disruption of Trp3-inositol trisphos-phate receptor association. J Biol Chem 276:42401–44248.

40. Zhang SL, et al. (2006)Genome-wide RNAi screen of Ca2� influx identifies genes thatregulate Ca2� release-activated Ca2� channel activity. Proc Natl Acad Sci USA 103:9357–9362.,

41. Peinelt C, et al. (2006) Amplification of CRAC current by STIM1 and CRACM1 (Orai1).Nat Cell Biol 8:771–773.

42. Soboloff J, et al. (2006) Orai1 and STIM reconstitute store-operated calcium channelfunction. J Biol Chem 281:20661–20665.

43. Mercer JC, et al. (2006) Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1. J Biol Chem281:24979–24990.

44. Zhu X, et al. (1996) trp, a Novel Mammalian Gene Family Essential for Agonist-activatedCapacitative Ca2� Entry. Cell 85:661–671.

3206 � www.pnas.org�cgi�doi�10.1073�pnas.0813346106 Liao et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

, 202

1