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Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1-forming Ca2+ Channels

2008; Elsevier BV; Volume: 283; Issue: 37 Linguagem: Inglês

10.1074/jbc.m802904200

1083-351X

Isaac Jardín, José J. López, Ginés M. Salido, Juan A. Rosado,

Neurobiology and Insect Physiology Research

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca2+ selective ICRAC and relatively Ca2+ selective ISOC, respectively. STIM1 has been proposed to communicate the Ca2+ content of the intracellular Ca2+ stores to the plasma membrane store-operated Ca2+ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca2+ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca2+. In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca2+ permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca2+ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca2+ entry. Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca2+ selective ICRAC and relatively Ca2+ selective ISOC, respectively. STIM1 has been proposed to communicate the Ca2+ content of the intracellular Ca2+ stores to the plasma membrane store-operated Ca2+ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca2+ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca2+. In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca2+ permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca2+ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca2+ entry. Store-operated calcium entry (SOCE), 4The abbreviations used are: SOCEstore-operated calcium entry[Ca2+]iintracellular free calcium concentrationCRACCa2+ release-activated currentDAGdiacylglycerolERendoplasmic reticulumHBSHEPES-buffered salinehTRPC1human canonical TRP1IP3Rinositol 1,4,5-trisphosphate receptorJPjasplakinolideOAG1-oleoyl-2-acetyl-sn-glycerolPBSphosphate-buffered salinePLCphospholipase CPMplasma membraneSOCstore-operated Ca2+STIM1stromal interaction molecule 1TGthapsigarginTRPtransient receptor potentialDMSOdimethyl sulfoxideJPjasplakinolide. 4The abbreviations used are: SOCEstore-operated calcium entry[Ca2+]iintracellular free calcium concentrationCRACCa2+ release-activated currentDAGdiacylglycerolERendoplasmic reticulumHBSHEPES-buffered salinehTRPC1human canonical TRP1IP3Rinositol 1,4,5-trisphosphate receptorJPjasplakinolideOAG1-oleoyl-2-acetyl-sn-glycerolPBSphosphate-buffered salinePLCphospholipase CPMplasma membraneSOCstore-operated Ca2+STIM1stromal interaction molecule 1TGthapsigarginTRPtransient receptor potentialDMSOdimethyl sulfoxideJPjasplakinolide. a process controlled by the filling state of the intracellular Ca2+ stores (1Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar), is a major mechanism for Ca2+ influx in non-excitable cells. Because SOCE was first proposed two decades ago, many studies have been devoted to the identification of the mechanisms that communicate the Ca2+ stores with the plasma membrane (PM) channels, as well as the nature of store-operated Ca2+ (SOC) channels. The first identified and best-characterized store-operated current is ICRAC, but a number of other SOC currents activated by Ca2+ store depletion have also been described (2Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1771) Google Scholar). store-operated calcium entry intracellular free calcium concentration Ca2+ release-activated current diacylglycerol endoplasmic reticulum HEPES-buffered saline human canonical TRP1 inositol 1,4,5-trisphosphate receptor jasplakinolide 1-oleoyl-2-acetyl-sn-glycerol phosphate-buffered saline phospholipase C plasma membrane store-operated Ca2+ stromal interaction molecule 1 thapsigargin transient receptor potential dimethyl sulfoxide jasplakinolide. store-operated calcium entry intracellular free calcium concentration Ca2+ release-activated current diacylglycerol endoplasmic reticulum HEPES-buffered saline human canonical TRP1 inositol 1,4,5-trisphosphate receptor jasplakinolide 1-oleoyl-2-acetyl-sn-glycerol phosphate-buffered saline phospholipase C plasma membrane store-operated Ca2+ stromal interaction molecule 1 thapsigargin transient receptor potential dimethyl sulfoxide jasplakinolide. The discovery of mammalian homologues of the Drosophila transient receptor potential (TRP) channel proteins has focused attention on TRP channels, especially the canonical TRP (TRPC) channels, as candidates for the conduction of SOCE (3Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar, 4Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (345) Google Scholar, 5Rosado J.A. Brownlow S.L. Sage S.O. J. Biol. Chem. 2002; 277: 42157-42163Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and a functional coupling between several TRPCs and IP3 receptor isoforms (IP3Rs) has been demonstrated in transfected cells and cells naturally expressing TRPC proteins (4Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (345) Google Scholar, 6Yuan J.P. Kiselyov K. Shin D.M. Chen J. Shcheynikov N. Kang S.H. Dehoff M.H. Schwarz M.K. Seeburg P.H. Muallem S. Worley P.F. Cell. 2003; 114: 777-789Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 7Jardin I. López J.J. Salido G.M. Rosado J.A. Cell Signal. 2008; 20: 737-747Crossref PubMed Scopus (38) Google Scholar). The recent identification of proteins STIM1 and Orai1 has shed new light on the nature and regulation of SOC channels. Orai1 (also named CRACM1 for Ca2+ release-activated current (CRAC) modulator) has been proposed to form the pore of the channel mediating ICRAC (8Vig M. Beck A. Billingsley J.M. Lis A. Parvez S. Peinelt C. Koomoa D.L. Soboloff J. Gill D.L. Fleig A. Kinet J.P. Penner R. Science. 2006; 312: 1220-1223Crossref PubMed Scopus (1126) Google Scholar). The involvement of Orai1 in ICRAC was identified by gene mapping in patients with hereditary severe combined immune deficiency syndrome attributed to loss of ICRAC (9Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. Nature. 2006; 441: 179-185Crossref PubMed Scopus (1805) Google Scholar, 10Zhang S.L. Yeromin A.V. Zhang X.H. Yu Y. Safrina O. Penna A. Roos J. Stauderman K.A. Cahalan M.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9357-9362Crossref PubMed Scopus (731) Google Scholar). Orai1 has been demonstrated to form multimeric ion-channel complexes in the PM (11Vig M. Beck A. Billingsley J.M. Lis A. Parvez S. Peinelt C. Koomoa D.L. Soboloff J. Gill D.L. Fleig A. Kinet J.P. Pender R. Curr. Biol. 2006; 16: 2073-2079Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). The channel formed by Orai1 has been reported to be regulated by Ca2+ store depletion with the participation of the intraluminal Ca2+ sensor, stromal interaction molecule 1 (STIM1), a protein that has recently been presented as a messenger linking the endoplasmic reticulum (ER) to PM Ca2+ channels. STIM1 is a Ca2+-binding protein located mainly in the ER membrane with a single transmembrane region and a EF-hand domain in the NH2 terminus located in the lumen of the ER (12Liou J. Kim M.L. Heo W.D. Jones T.J. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar), that might, therefore, function as a Ca2+ sensor in the ER (13Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Velicelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1485) Google Scholar, 14Putney Jr., J.W. J. Cell Biol. 2005; 169: 381-382Crossref PubMed Scopus (144) Google Scholar). Knockdown of STIM1 by RNA interference or functional knockdown of STIM1 by electrotransjection of neutralizing antibodies reduces SOCE in HEK293, HeLa, and Jurkat T cells and platelets (12Liou J. Kim M.L. Heo W.D. Jones T.J. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar, 13Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Velicelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1485) Google Scholar, 15López J.J. Salido G.M. Pariente J.A. Rosado J.A. J. Biol. Chem. 2006; 281: 28254-28264Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and ICRAC in Jurkat T cells (13Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Velicelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1485) Google Scholar). In support of the role of STIM1 in SOCE, mutation of the Ca2+ binding EF-hand domain of STIM1 resulted in constitutive SOC channel activation without any detectable change in the content of the Ca2+ stores (16Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1113) Google Scholar). The cytoplasmic COOH-terminal domain of STIM1 has been suggested to interact with the NH2 terminus of Orai1, facilitating the Orai1-STIM1 interactions required for the activation of ICRAC (17Stathopulos P.B. Li G.Y. Pleven M.J. Ames J.B. Ikura M. J. Biol. Chem. 2006; 281: 35855-35862Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). In addition, hTRPC1 has been presented as an essential component of the SOC channels. Heteromeric interactions of TRPC1 with other TRPCs have been reported to lead to the generation of SOC channels with different biophysical properties (18Ambudkar I.S. Ong H.L. Liu X. Bandyopadhyay B. Cheng K.T. Cell Calcium. 2007; 42: 213-223Crossref PubMed Scopus (203) Google Scholar). In human platelets, hTRPC1 forms a complex hTRPC6, the type II IP3 receptor and SERCA3 activated by depletion of the intracellular Ca2+ stores (19Redondo P.C. Jardin I. Lopez J.J. Salido G.M. Rosado J.A. Biochim. Biophys. Acta. 2008; 1783: 1163-1176Crossref PubMed Scopus (56) Google Scholar). In addition, a recent study has reported that TRPC1 associates with STIM1 and Orai1 in culture cells to form a ternary complex that is important for the formation of the SOC channel (20Ong H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Orai1 has been shown to confer to TRPCs STIM1-mediated store-operated sensitivity (21Liao Y. Erxleben C. Yildirim E. Abramowitz J. Armstrong D.L. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4682-4687Crossref PubMed Scopus (256) Google Scholar); however, it remains unclear whether hTRPC1 interacts directly with STIM1 or through Orai1 and whether the heteromeric Orai1-TRPC1 interaction forms a channel sensitive to store depletion independently of STIM1. In the present study we have investigated the interaction of endogenously expressed Orai1 with STIM1 and hTRPC1 at resting conditions and upon store depletion either by pharmacological tools or with the physiological agonist thrombin. In addition, we have investigated the role of the STIM1-Orai1 interaction on SOCE and the mode of activation of hTRPC1-forming channels. Our results indicate that Ca2+ store depletion stimulates rapid and transient interaction between Orai1 and both, STIM1 and hTRPC1. Electrotransjection with anti-Orai1 COOH terminus antibody or treatment with jasplakinolide (JP) prevented the interaction of STIM1 with Orai1 and hTRPC1, reduced SOCE, and changed the mode of activation of hTRPC1-forming channels. Materials—Fura-2 acetoxymethyl ester (fura-2/AM), JP, and calcein/AM were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin, thrombin, thapsigargin (TG), leupeptin, benzamidine, phenylmethylsulfonyl fluoride, SDS, anti-actin antibody, rabbit anti-Orai antibody (COOH-terminal), and bovine serum albumin were from Sigma. Anti-STIM1 antibody was from BD Transduction Laboratories (Franklin Lakes, NJ). Ionomycin and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were from Calbiochem (Madrid, Spain). Anti-hTRPC1 polyclonal antibody was obtained from Alomone Laboratories (Jerusalem, Israel). Horseradish peroxidase-conjugated goat anti-rabbit IgG antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated ovine anti-mouse IgG antibody (NA931) and hyperfilm ECL were from Amersham Biosciences. Protein A-agarose was from Upstate Biotechnology Inc. (Madrid, Spain). Enhanced chemiluminescence detection reagents were from Pierce. All other reagents were of analytical grade. Platelet Preparation—Platelet suspensions were prepared as previously described (22López J.J. Camello-Almaraz C. Pariente J.A. Salido G.M. Rosado J.A. Biochem. J. 2005; 390: 243-252Crossref PubMed Scopus (105) Google Scholar) as approved by Local Ethical Committees and in accordance with the Declaration of Helsinki. Briefly, blood was obtained from healthy drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anti-coagulant containing (in mm): 85 sodium citrate, 78 citric acid, and 111 d-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 × g and aspirin (100 μm) and apyrase (40 μg/ml) were added. Platelets were then collected by centrifugation at 350 × g for 20 min and resuspended in HEPES-buffered saline (HBS), pH 7.45, containing (in mm): 145 NaCl, 10 HEPES, 10 d-glucose, 5 KCl, 1 MgSO4 and supplemented with 0.1% bovine serum albumin and 40 μg/ml apyrase. Cell viability was assessed using calcein and trypan blue. For calcein loading, platelets were incubated for 30 min with 5 μm calcein-AM at 37 °C, centrifuged, and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2-ml aliquots using a Cary Eclipse spectrophotometer (Varian Ltd., Madrid, Spain). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. The results obtained with calcein were confirmed using the trypan blue exclusion technique. 95% of platelets were viable in our preparations. Measurement of Intracellular Free Calcium Concentration ([Ca2+]i)—Human platelets were loaded with fura-2 by incubation with 2 μm fura-2/AM for 45 min at 37 °C. Fluorescence was recorded from 2-ml aliquots of magnetically stirred cellular suspension (2 × 108 platelets/ml) at 37 °C using a Cary Eclipse spectrophotometer (Varian Ltd.) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca2+]i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to a established method (23Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Ca2+ entry was estimated using the integral of the rise in [Ca2+]i for 2.5 min after addition of CaCl2 (22López J.J. Camello-Almaraz C. Pariente J.A. Salido G.M. Rosado J.A. Biochem. J. 2005; 390: 243-252Crossref PubMed Scopus (105) Google Scholar). OAG-induced Ca2+ entry was estimated using the integral of the rise in [Ca2+]i for 2.5 min after addition of OAG in a medium containing 1 mm Ca2+. Ca2+ entry was corrected by subtraction of the [Ca2+]i elevation due to leakage of the indicator or leak Ca2+ entry after the addition of DMSO (the vehicle of TG and OAG). Ca2+ release by TG was estimated using the integral of the rise in [Ca2+]i for 3 min after the addition of the agent (22López J.J. Camello-Almaraz C. Pariente J.A. Salido G.M. Rosado J.A. Biochem. J. 2005; 390: 243-252Crossref PubMed Scopus (105) Google Scholar). Ca2+ entry and release are expressed as nm·s, as previously described (24Rosado J.A. Sage S.O. Biochem. J. 2000; 347: 183-192Crossref PubMed Scopus (92) Google Scholar). Immunoprecipitation and Western Blotting—The immunoprecipitation and Western blotting were performed as described previously (15López J.J. Salido G.M. Pariente J.A. Rosado J.A. J. Biol. Chem. 2006; 281: 28254-28264Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Briefly, 500-μl aliquots of platelet suspension (2 × 109 cell/ml) were lysed with an equal volume of RIPA buffer, pH 7.2, containing 316 mm NaCl, 20 mm Tris, 2 mm EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mm Na3VO4, 2 mm phenylmethylsulfonyl fluoride, 100 μg/ml leupeptin, and 10 mm benzamidine. Aliquots of platelet lysates (1 ml) were immunoprecipitated by incubation with 2 μg of anti-Orai1 antibody and 25 μl of protein A-agarose overnight at 4 °C on a rocking platform. The immunoprecipitates were resolved by 10% SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of STIM1, hTRPC1, and Orai1 was achieved using the anti-STIM1 antibody diluted 1:250 in TBST for 2 h, the anti-hTRPC1 antibody diluted 1:200 in TBST for 1 h, and the anti-Orai1 antibody diluted 1:1000 in TBST for 1.5 h, respectively. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 45 min with horseradish peroxidase-conjugated ovine anti-mouse IgG antibody or horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody diluted 1:10,000 in TBST and then exposed to enhanced chemiluminescence reagents for 4 min. Blots were then exposed to photographic films. The density of bands on the film was measured using scanning densitometry. Reversible Electroporation Procedure—The platelet suspension was transferred to an electroporation chamber containing antibodies at a final concentration of 2 μg/ml, and the antibodies were transjected according to published methods (15López J.J. Salido G.M. Pariente J.A. Rosado J.A. J. Biol. Chem. 2006; 281: 28254-28264Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Reversible electropermeabilization was performed at 4 kV/cm at a setting of 25-microfarad capacitance and was achieved by 7 pulses using a Bio-Rad Gene Pulser Xcell Electroporation System (Bio-Rad). Following electroporation, platelets were incubated with antibodies for an additional 60 min at 37 °C and centrifuged at 350 × g for 20 min and resuspended in HBS prior to the experiments. Statistical Analysis—Analysis of statistical significance was performed using Student's t test. p < 0.05 was considered to be significant for a difference. Orai1 Co-immunoprecipitates with hTRPC1 and STIM1 in Human Platelets—Platelets have been shown to endogenously express hTRPC1 channel in the PM (5Rosado J.A. Brownlow S.L. Sage S.O. J. Biol. Chem. 2002; 277: 42157-42163Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), and a functional interaction between STIM1 in the Ca2+ stores and hTRPC1 has been reported to account for the activation of SOCE in these cells (15López J.J. Salido G.M. Pariente J.A. Rosado J.A. J. Biol. Chem. 2006; 281: 28254-28264Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We have now investigated the association between hTRPC1 and Orai1 by looking for co-immunoprecipitation from platelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using control platelets and platelets treated in the absence of extracellular Ca2+ (100 μm EGTA was added to the medium) for different periods of time (from 10 to 60 s) with inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) TG (1 μm) plus a low concentration of ionomycin (50 nm), to induce extensive depletion of the intracellular stores in platelets (25Juska A. Redondo P.C. Rosado J.A. Salido G.M. Biochem. Biophys. Res. Commun. 2005; 334: 779-786Crossref PubMed Scopus (17) Google Scholar). After immunoprecipitation with anti-hTRPC1 or anti-Orai1 antibodies, Western blotting revealed the presence of Orai1 in samples from resting platelets. The specificity of the hTRPC1 antibody was tested with the anti-TRPC1 antibody T1E3, which has been shown to be a specific tool in the investigation of mammalian TRPC1 proteins (5Rosado J.A. Brownlow S.L. Sage S.O. J. Biol. Chem. 2002; 277: 42157-42163Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 26Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Crossref PubMed Scopus (292) Google Scholar). We found that treatment with TG + ionomycin increased the association between Orai1 and hTRPC1 in a time-dependent manner, reaching a maximal effect after 30 s of platelet stimulation (Fig. 1A, upper panel; n = 6). Similar results were observed when cells were stimulated with the physiological agonist thrombin (1 units/ml) in a Ca2+-free medium (100 μm EGTA was added at the time of experiment). Thrombin increased coimmunoprecipitation between Orai1 and hTRPC1 in a time-dependent manner, reaching a maximum after 10 s of stimulation with the agonist (Fig. 1B, upper panel; n = 6). Western blotting of the same membranes with the antibody used for immunoprecipitation confirmed similar protein content in all lanes (Fig. 1, lower panels). Furthermore, we have explored the association between STIM1 and Orai1 by looking for co-immunoprecipitation from platelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using control platelets and platelets treated in a Ca2+-free medium (100 μm EGTA added) for different periods of time (from 10 to 60 s) with TG (1 μm) and ionomycin (50 nm). Our results indicate that treatment with TG + ionomycin increased the association between Orai1 and STIM1 in a time-dependent manner, reaching a maximal effect after 10 s of platelet stimulation (Fig. 2A, upper panel; n = 6). Similar results were observed when cells were stimulated with thrombin, which increased co-immunoprecipitation between Orai1 and STIM1 in a time-dependent manner, reaching a maximum after 30 s of stimulation with the agonist (Fig. 2B, upper panel; n = 6). Western blotting of the same membranes with the antibody used for immunoprecipitation confirmed similar protein content in all lanes (Fig. 2, lower panels). Our observations, showing an enhanced association of Orai1 with hTRPC1 and STIM1 in response to depletion of the intracellular Ca2+ stores or the physiological agonist thrombin suggest that the STIM1-Orai1-hTRPC1 ternary complex might be important for the mediation of SOCE in these cells. Inhibition of Store Depletion-evoked Interaction between STIM1 and hTRPC1 by Electrotransjection with Anti-Orai1 C-terminal Antibody—The amino acid sequence 288–301 of human Orai1 recognized by the anti-Orai1 antibody used is located in the cytosolic COOH-terminal region of Orai1, which has been shown to be essential for the interaction of Orai1 with STIM1 (27Muik M. Frischauf I. Derler I. Fahrner M. Bergsmann J. Eder P. Schindl R. Hesch C. Polzinger B. Fritsch R. Kahr H. Madl J. Gruber H. Groschner K. Romanin C. J. Biol. Chem. 2008; 283: 8014-8022Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Since Orai1 has been proposed to mediate the communication between STIM1 and hTRPC1 (21Liao Y. Erxleben C. Yildirim E. Abramowitz J. Armstrong D.L. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4682-4687Crossref PubMed Scopus (256) Google Scholar) we have investigated whether the anti-Orai1 antibody, which is directed to the COOH-terminal region, could block the interaction between STIM1 and hTRPC1. To assess this possibility the anti-Orai1 antibody was introduced into platelets using an electropermeabilization technique. Electroporation can be used successfully for transferring antibodies into cells while maintaining the physiological integrity of the cells (15López J.J. Salido G.M. Pariente J.A. Rosado J.A. J. Biol. Chem. 2006; 281: 28254-28264Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 28Chakrabarti R. Wylie D.E. Schuster S.M. J. Biol. Chem. 1989; 264: 15494-15500Abstract Full Text PDF PubMed Google Scholar, 29Dhar A. Shukla S.D. J. Biol. Chem. 1994; 269: 9123-9127Abstract Full Text PDF PubMed Google Scholar). Human platelets were reversibly electroporated as described under "Experimental Procedures." The presence of this antibody inside platelets was confirmed in samples from platelets electropermeabilized and incubated with 1 μg/ml of either anti-Orai1 antibody or rabbit IgG, of the same nature of the anti-Orai1 antibody used, by immunoprecipitation without adding any additional antibody and subsequent Western blotting with the anti-Orai1 antibody. As shown in Fig. 3A, Orai1 was clearly detected in cells that had been previously electropermeabilized and incubated with anti-Orai1 antibody and not in cells incubated with rabbit IgG. Electropermeabilization allowed the anti-Orai1 antibody to enter the cells and immunoprecipitate native Orai1, which was then detected by Western blotting. To further investigate whether reversible electroporation might induce loss of proteins of the size of Orai1 (∼45 kDa) (30Gwack Y. Srikanth S. Feske S. Cruz-Guilloty F. Oh-hora M. Neems D.S. Hogan P.G. Rao A. J. Biol. Chem. 2007; 282: 16232-16243Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) we investigated the presence of actin (42 kDa) in electroporated and non-electroporated platelets. As shown in Fig. 3B, the amount of actin detected by Western blotting in electroporated platelets was not significantly smaller than that detected in non-electroporated platelets. Altogether, these findings confirm the efficacy of the electrotransjection and that the amount of Orai1 detected was not modified by treatment with TG + ionomycin for 30 s (Fig. 3A, top panel). As shown in Fig. 3C, interaction between STIM1 and hTRPC1 was abolished in platelets electrotransjected with 1 μg/ml anti-Orai1 antibody (upper panel, third and fourth lanes; p < 0.001; n = 6) compared with platelets electrotransjected with 1 μg/ml rabbit IgG, as detected by immunoprecipitation of cell lysates with the anti-STIM1 antibody followed by Western blotting with anti-hTRPC1 antibody. Reprobing of the same membranes with anti-STIM1 antibody confirmed a similar protein loading in all lanes (Fig. 3C, lower panel). We found that electrotransjection of the anti-Orai1 antibody inhibits TG + ionomycin-induced Orai1-STIM1 co-immunoprecipitation by performing immunoprecipitation with the transjected anti-Orai1 antibody (no additional antibodies were added for immunoprecipitation after transjection of anti-Orai1 antibody into cells) followed by Western blotting with the anti-STIM1 antibody (data not shown). These findings were not observed when platelets were electrotransjected with rabbit IgG (data not shown). These findings suggest that the amino acid sequence recognized by the anti-Orai1 antibody is essential for the interaction of STIM1 and hTRPC1, and blockade of this interaction might impair the function of the STIM1-Orai1-hTRPC1 ternary complex. Impairment of the Interaction between STIM1 and hTRPC1 Changes the Behavior of hTRPC1-forming Channels from Capacitative to Non-capacitative Channel—We have further investigated whether the anti-Orai1 antibody could affect SOCE in these cells. To assess this issue, the anti-Orai1 antibody was electrotransjected into platelets, followed by depletion of the intracellular Ca2+ stores using TG (200 nm) to activate SOCE. Before the measurement of [Ca2+]i platelets were maintained in a medium containing 200 μm CaCl2, to avoid premature depletion of the stores. At the time of the experiment 250 μm EGTA was added to perform the studies in a Ca2+-free medium. In platelets electrotransjected with rabbit IgG (Fig. 4A), TG evoked a prolonged elevation of [Ca2+]i, due to leakage of Ca2+ from intracellular stores (the integral for 3 min of the rise in [Ca2+]i after the addition of TG was 238 ± 73 nm·s; Fig. 4A, rabbit IgG: Control). Subsequent addition of Ca2+ (1 mm) to the external medium induced a sustained increase in [Ca2+]i, indicative of SOCE (the integral of the rise in [Ca2+]i after the addition of CaCl2 was 926 ± 137 nm·s; Fig. 4A, rabbit IgG: Control). To assess the involvement of hTRPC1 in TG-evoked SOCE we incubated cells for 30 min with 15 μm anti-