TRPC1 Is Required for Functional Store-operated Ca2+ Channels
2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês
10.1074/jbc.m213271200
ISSN1083-351X
AutoresXibao Liu, Brij B. Singh, Indu S. Ambudkar,
Tópico(s)Magnesium in Health and Disease
ResumoThe exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557–620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, ISOC, were not altered. Expression of TRPC1Δ567–793, but not TRPC1Δ664–793, induced a similar decrease in SOCC activity. Furthermore, TRPC1Δ567–793 was co-immunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6 region (Asp → Asn and Glu → Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left shift in E rev. Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx. The exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557–620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, ISOC, were not altered. Expression of TRPC1Δ567–793, but not TRPC1Δ664–793, induced a similar decrease in SOCC activity. Furthermore, TRPC1Δ567–793 was co-immunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6 region (Asp → Asn and Glu → Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left shift in E rev. Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx. phosphatidylinositol bisphosphate green fluorescent protein store-operated calcium influx channel amino acids immunoprecipitation hemagglutinin thapsigargin carbachol Ca2+ release activated Ca2+channel transient receptor potential protein TRP canonical family TRP vanalloid family TRP melastatin family Activation of cell surface receptors, which are coupled to inositol lipid signaling, results in phosphatidylinositol bisphosphate (PIP2)1hydrolysis, generation of diacylglycerol and inositol 1,4,5-trisphosphate, release of Ca2+ from internal Ca2+ stores, and activation of plasma membrane Ca2+ influx channels (1Putney Jr., J.W. Pharmacol. Ther. 1990; 48: 427-434Google Scholar, 2Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). Two types of plasma membrane channels have been associated with this signaling mechanism; store-independent channels that appear to be activated as a result of PIP2 hydrolysis and store-operated Ca2+channels, SOCC, that are activated primarily in response to depletion of Ca2+ from the internal Ca2+ stores (3Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Google Scholar, 4Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Google Scholar). However, the mechanism(s) underlying activation and gating of these plasma membrane calcium channels as well as their molecular composition have remained elusive for more than a decade (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Google Scholar, 6Putney Jr., J.W. Adv. Pharmacol. 1991; 22: 251-269Google Scholar, 7Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Google Scholar). The most well characterized SOCC is the Ca2+ release activated Ca2+ channel (CRAC) that mediates the store-operated current ICRAC in mast cells and lymphocytes (8Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Google Scholar, 9Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Google Scholar). This channel has been reported to have a very high selectivity for Ca2+ and unique inactivation properties. SOCCs have also been described in a number of other cell types. Many of these display characteristics different from CRAC and from each other (7Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Google Scholar, 8Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Google Scholar). Based on the distinct activities seen in different cell types, it is likely that the molecular composition, or regulation, of their SOCCs is different.Members of the TRP superfamily of putative ion channel proteins have been suggested as components of SOCCs (10Zhu X. Jiang M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Google Scholar). Although it is possible that some TRPVs or TRPMs might form SOCCs (11Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Google Scholar, 12Schindl R. Kahr H. Graz I. Groschner K. Romanin C. J. Biol. Chem. 2002; 277: 26950-26958Google Scholar, 13Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G. Bindels R.J. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Google Scholar), there are sufficient data to demonstrate that the TRPC family is involved in agonist-stimulated Ca2+ signaling (3Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Google Scholar, 4Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Google Scholar,14Montell C. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1)Google Scholar). It is now clear that some TRPCs, e.g. TRPC1 and TRPC4, allow plasma membrane Ca2+ influx in response to depletion of the internal Ca2+ store. Others, like TRPC3 and TRPC6, appear to be activated by PIP2 hydrolysis in a number of cell types. Compared with the other TRPCs, TRPC1 displays a relatively wider tissue distribution (15Zhu X. Chu P.B. Peyton M. Birnbaumer L. FEBS Lett. 1995; 373: 193-198Google Scholar, 16Wang W. O'Connell B. Dykeman R. Sakai T. Delporte C. Swaim W. Zhu X. Birnbaumer L. Ambudkar I.S. Am. J. Physiol. 1999; 276: 969-979Google Scholar). Furthermore, it is endogenously present and suggested to be involved in SOCE in several cell types as follows: salivary gland cells (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar), endothelial cells (19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar), vascular smooth muscle cells (21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar), and DT 40 cells (22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar) based on the decrease in SOCE induced by transfection with antisenseTRPC1 (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar), addition of the TRPC1 antibody extracellularly (20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar, 21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar), or knock-out of the TRPC1gene (22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar). Although these previous studies provide convincing evidence that TRPC1 has a critical role in SOCE, they do not elucidate whether TRPC1 serves as an essential regulatory subunit that is required for the assembly or regulation of the SOCCs in these cells or contributes more directly to the channel function per se,i.e. as a component of the pore-forming unit of the channel.We have previously suggested (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar) TRPC1 as a candidate for store-operated Ca2+ entry mechanism in the human submandibular gland cell line, HSG. We have also reported that the TRPC1 C terminus mediates Ca2+-dependent feedback regulation of SOCE in HSG cells (18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar, 23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar). To elucidate whether TRPC1 is a regulatory subunit of SOCC or contributes more directly to SOCC-mediated Ca2+ influx, here we have measured SOCE and SOCC activity in control HSG cells and in cells overexpressing either TRPC1 or TRPC1 with mutations in the proposed pore region. The data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx.DISCUSSIONPrevious studies (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar, 19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar, 21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar, 22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar, 23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) from our laboratory, and others, provide convincing evidence that TRPC1 has a critical role in SOCE. Based on the presently available data two possible roles can be assigned to TRPC1: (i) it is a regulatory subunit of SOCC that is required for its activation or assembly, and (ii) it is a component of the SOCC pore that directly contributes to SOCC-mediated Ca2+ influx. The data presented above suggest that TRPC1 is a functional component of an SOCC in HSG cells that contributes to Ca2+ influx in response to stimulation by either agonist or Tg. We have shown that the characteristics of SOCC activity generated by expression of TRPC1 in HSG cells are similar to the endogenous SOCC activity in these cells. Consistent with this, expression of TRPC1 in HSG cells induces an increase in the amplitude of the SOCC-mediated current, ISOC, without altering its characteristics (23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) (Fig. 4). We have also shown here and previously (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar) that endogenous TRPC1 in HSG cells is essential for SOCC activity. Tg- or CCh-stimulated SOCC activity, ISOC, and Ca2+ influx, as well as levels of endogenous TRPC1 in HSG cells are decreased by 80% or more in cells transfected with antisense-TRPC1. More significant is our finding that acidic amino acid residues in the proposed pore region of TRPC1 contribute to SOCE. We have shown that expression of TRPC1 with simultaneous mutations (Asp → Asn and Glu → Gln) of seven acidic aa residues in the proposed pore region suppressed endogenous SOCC activity in more than 80% of the cells. In the few cells expressing this mutant where channel activity was detected, there was an apparent decrease in the Ca2+ conductance. Importantly, the amplitude of ISOC in these cells was decreased, and there was a left shift in the reversal potential, suggesting a decrease in the Ca2+ permeability of the channel. Interestingly, the Na+ current mediated by Mut-pore was similar to that seen in TRPC1-cells. Thus, mutations in the acidic aa residues in the pore region do not appear to decrease SOCC activity per se. We have also demonstrated that two of these seven acidic amino acid residues, Glu-576 and Asp-581, are involved in SOCC-mediated Ca2+ influx. Substitution of either residue, E576K or D581K, induced a suppression of Tg-stimulated SOCE and ISOC. Notably, Glu-576 and Glu-615 are conserved in TRPC1, TRPC4, and TRPC5, although Asp-581 is present only in TRPC1. Our data also rule out the possibility that a decrease in SOCC activity in cells expressing mutant TRPC1 proteins is due to differences in protein expression or altered localization. In aggregate, these data support the proposal that TRPC1 is a component of the functional (i.e. pore-forming) unit of SOCC, rather than an associated regulatory protein. Although it is possible that a single mutation in an extracellular domain of TRPC1 might alter its interaction with an as yet unknown SOCC protein and lead to a decrease in Ca2+influx via the channel, it is highly unlikely.Previous studies have shown that Asp → Asn substitution in the pore region of TRPV4 decreased Ca2+ influx, although Asp → Lys substitutions completely blocked channel function (29Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Droogmans G. Bindels R.J. J. Biol. Chem. 2001; 276: 1020-1025Google Scholar). Substitution of conserved hydrophobic residues (LFW) in the TRPC6 pore region eliminated TRPC6-generated channel activity in HEK293 cells (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar). However, neither of these channels appear to be involved in SOCE. While this manuscript was in preparation, it was reported (31Cui J. Bian J.-S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Google Scholar) that expression of TRPV6 with substitution of FEL to AAA in the proposed pore region induced suppression of endogenous ICRAC in Jurkat cells. However, it is not clear whether the endogenous TRPV6 in Jurkat cells is required for ICRAC. Furthermore, TRPV6 displayed both store-operated and store-independent activities. The data we have presented above are significant because they demonstrate the following: (i) endogenous TRPC1 in HSG cells is required for the endogenous SOCE detected in these cells; (ii) expressed TRPC1 is regulated by store depletion and is not spontaneously active; and (iii) acidic aa residues in the pore region of TRPC1 contribute to SOCE.Although presently we do not have evidence for homo-multimerization of endogenous TRPC1 in HSG cells, we have shown that the expressed mutant TRPC1 is immunoprecipitated with endogenous TRPC1. This result has major implications because it suggests that TRPC1 monomers interact with each other to form SOCC. Our data are consistent with reports suggesting that Drosophila TRP and TRPC1β homomultimerize via N-terminal interactions (32Engelke M. Friedrich O. Budde P. Schafer C. Niemann U. Zitt C. Jungling E. Rocks O. Luckhoff A. Frey J. FEBS Lett. 2002; 17 (193–199): 523Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar). We suggest that endogenous or exogenously expressed TRPC1 monomers associate with each other to form functional SOCCs. Thus, when full-length TRPC1 is expressed channel activity is increased. When mutant TRPC1s are overexpressed relative to the endogenous protein, the probability of mutant TRPC1 monomers associating with each other or with endogenous TRPC1 monomers is relatively high. As a result, aberrant SOCCs are formed which have decreased permeability for calcium. In contrast, in cells transfected with antisense TRPC1 or expressing TRPC1Δ567–793, where either depletion of TRPC1 or the pore region is deleted, respectively, there is a reduction in the total number of functional channels rather than a change in the channel properties. However, other proteins, including other TRPCs, might be co-assembled with endogenous TRPC1 and required for functional SOCC in HSG cells. Studies using heterologous expression have shown that TRPC1 interacts with TRPC3 (33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar), TRPC6 (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar), and TRPC4 and TRPC5 (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). The latter study also showed that endogenous heteromers of either TRPC1 and TRPC4 or TRPC1 and TRPC5 were co-immunoprecipitated from rat brain. TRPC1 expression altered the currents generated when either TRPC5 or TRPC4 was expressed alone, and furthermore, these currents were not activated by store depletion. Although it is unclear why TRPC1 forms different types of channels in different cells, it is important to note that the characteristics of the channel formed by heteromeric TRPC1 channels (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) appear to be considerably different from those of ISOCmeasured in HSG cells. Further studies will be required to determine which TRPC proteins are endogenously expressed in HSG cells and interact with endogenous TRPC1.In conclusion, the data presented here suggest that TRPC1 is a component of the functional (pore-forming) unit of SOCC in HSG cells. However, these data do not rule out the possibility that SOCC might be a heteromer of TRPC1 with other TRPCs (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Google Scholar, 30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar, 35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) or with other as yet unknown protein(s). We have shown earlier that in HSG cells TRPC1, like the Drosophila TRP (14Montell C. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1)Google Scholar, 36Li H.S. Montell C. J. Cell Biol. 2000; 150: 1411-1422Google Scholar), is assembled in a supramolecular protein complex with key proteins involved in the Ca2+ signaling cascade that leads to SOCC activation (26Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2002; 275: 11934-11942Google Scholar). We suggest that SOCC activity in any cell type will depend not only on the proteins that constitute its pore-forming unit but also other regulatory proteins that might affect its function, assembly, or localization. It will be important to determine whether differences in the molecular composition of the channel per se, or its regulation, account for the large variation in the characteristics of SOCCs seen in different cell types. Activation of cell surface receptors, which are coupled to inositol lipid signaling, results in phosphatidylinositol bisphosphate (PIP2)1hydrolysis, generation of diacylglycerol and inositol 1,4,5-trisphosphate, release of Ca2+ from internal Ca2+ stores, and activation of plasma membrane Ca2+ influx channels (1Putney Jr., J.W. Pharmacol. Ther. 1990; 48: 427-434Google Scholar, 2Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). Two types of plasma membrane channels have been associated with this signaling mechanism; store-independent channels that appear to be activated as a result of PIP2 hydrolysis and store-operated Ca2+channels, SOCC, that are activated primarily in response to depletion of Ca2+ from the internal Ca2+ stores (3Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Google Scholar, 4Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Google Scholar). However, the mechanism(s) underlying activation and gating of these plasma membrane calcium channels as well as their molecular composition have remained elusive for more than a decade (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Google Scholar, 6Putney Jr., J.W. Adv. Pharmacol. 1991; 22: 251-269Google Scholar, 7Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Google Scholar). The most well characterized SOCC is the Ca2+ release activated Ca2+ channel (CRAC) that mediates the store-operated current ICRAC in mast cells and lymphocytes (8Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Google Scholar, 9Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Google Scholar). This channel has been reported to have a very high selectivity for Ca2+ and unique inactivation properties. SOCCs have also been described in a number of other cell types. Many of these display characteristics different from CRAC and from each other (7Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Google Scholar, 8Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Google Scholar). Based on the distinct activities seen in different cell types, it is likely that the molecular composition, or regulation, of their SOCCs is different. Members of the TRP superfamily of putative ion channel proteins have been suggested as components of SOCCs (10Zhu X. Jiang M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Google Scholar). Although it is possible that some TRPVs or TRPMs might form SOCCs (11Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Google Scholar, 12Schindl R. Kahr H. Graz I. Groschner K. Romanin C. J. Biol. Chem. 2002; 277: 26950-26958Google Scholar, 13Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G. Bindels R.J. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Google Scholar), there are sufficient data to demonstrate that the TRPC family is involved in agonist-stimulated Ca2+ signaling (3Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Google Scholar, 4Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. 2002; 66: 243-264Google Scholar,14Montell C. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1)Google Scholar). It is now clear that some TRPCs, e.g. TRPC1 and TRPC4, allow plasma membrane Ca2+ influx in response to depletion of the internal Ca2+ store. Others, like TRPC3 and TRPC6, appear to be activated by PIP2 hydrolysis in a number of cell types. Compared with the other TRPCs, TRPC1 displays a relatively wider tissue distribution (15Zhu X. Chu P.B. Peyton M. Birnbaumer L. FEBS Lett. 1995; 373: 193-198Google Scholar, 16Wang W. O'Connell B. Dykeman R. Sakai T. Delporte C. Swaim W. Zhu X. Birnbaumer L. Ambudkar I.S. Am. J. Physiol. 1999; 276: 969-979Google Scholar). Furthermore, it is endogenously present and suggested to be involved in SOCE in several cell types as follows: salivary gland cells (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar), endothelial cells (19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar), vascular smooth muscle cells (21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar), and DT 40 cells (22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar) based on the decrease in SOCE induced by transfection with antisenseTRPC1 (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar), addition of the TRPC1 antibody extracellularly (20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar, 21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar), or knock-out of the TRPC1gene (22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar). Although these previous studies provide convincing evidence that TRPC1 has a critical role in SOCE, they do not elucidate whether TRPC1 serves as an essential regulatory subunit that is required for the assembly or regulation of the SOCCs in these cells or contributes more directly to the channel function per se,i.e. as a component of the pore-forming unit of the channel. We have previously suggested (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar) TRPC1 as a candidate for store-operated Ca2+ entry mechanism in the human submandibular gland cell line, HSG. We have also reported that the TRPC1 C terminus mediates Ca2+-dependent feedback regulation of SOCE in HSG cells (18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar, 23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar). To elucidate whether TRPC1 is a regulatory subunit of SOCC or contributes more directly to SOCC-mediated Ca2+ influx, here we have measured SOCE and SOCC activity in control HSG cells and in cells overexpressing either TRPC1 or TRPC1 with mutations in the proposed pore region. The data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx. DISCUSSIONPrevious studies (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar, 19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar, 21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar, 22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar, 23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) from our laboratory, and others, provide convincing evidence that TRPC1 has a critical role in SOCE. Based on the presently available data two possible roles can be assigned to TRPC1: (i) it is a regulatory subunit of SOCC that is required for its activation or assembly, and (ii) it is a component of the SOCC pore that directly contributes to SOCC-mediated Ca2+ influx. The data presented above suggest that TRPC1 is a functional component of an SOCC in HSG cells that contributes to Ca2+ influx in response to stimulation by either agonist or Tg. We have shown that the characteristics of SOCC activity generated by expression of TRPC1 in HSG cells are similar to the endogenous SOCC activity in these cells. Consistent with this, expression of TRPC1 in HSG cells induces an increase in the amplitude of the SOCC-mediated current, ISOC, without altering its characteristics (23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) (Fig. 4). We have also shown here and previously (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar) that endogenous TRPC1 in HSG cells is essential for SOCC activity. Tg- or CCh-stimulated SOCC activity, ISOC, and Ca2+ influx, as well as levels of endogenous TRPC1 in HSG cells are decreased by 80% or more in cells transfected with antisense-TRPC1. More significant is our finding that acidic amino acid residues in the proposed pore region of TRPC1 contribute to SOCE. We have shown that expression of TRPC1 with simultaneous mutations (Asp → Asn and Glu → Gln) of seven acidic aa residues in the proposed pore region suppressed endogenous SOCC activity in more than 80% of the cells. In the few cells expressing this mutant where channel activity was detected, there was an apparent decrease in the Ca2+ conductance. Importantly, the amplitude of ISOC in these cells was decreased, and there was a left shift in the reversal potential, suggesting a decrease in the Ca2+ permeability of the channel. Interestingly, the Na+ current mediated by Mut-pore was similar to that seen in TRPC1-cells. Thus, mutations in the acidic aa residues in the pore region do not appear to decrease SOCC activity per se. We have also demonstrated that two of these seven acidic amino acid residues, Glu-576 and Asp-581, are involved in SOCC-mediated Ca2+ influx. Substitution of either residue, E576K or D581K, induced a suppression of Tg-stimulated SOCE and ISOC. Notably, Glu-576 and Glu-615 are conserved in TRPC1, TRPC4, and TRPC5, although Asp-581 is present only in TRPC1. Our data also rule out the possibility that a decrease in SOCC activity in cells expressing mutant TRPC1 proteins is due to differences in protein expression or altered localization. In aggregate, these data support the proposal that TRPC1 is a component of the functional (i.e. pore-forming) unit of SOCC, rather than an associated regulatory protein. Although it is possible that a single mutation in an extracellular domain of TRPC1 might alter its interaction with an as yet unknown SOCC protein and lead to a decrease in Ca2+influx via the channel, it is highly unlikely.Previous studies have shown that Asp → Asn substitution in the pore region of TRPV4 decreased Ca2+ influx, although Asp → Lys substitutions completely blocked channel function (29Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Droogmans G. Bindels R.J. J. Biol. Chem. 2001; 276: 1020-1025Google Scholar). Substitution of conserved hydrophobic residues (LFW) in the TRPC6 pore region eliminated TRPC6-generated channel activity in HEK293 cells (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar). However, neither of these channels appear to be involved in SOCE. While this manuscript was in preparation, it was reported (31Cui J. Bian J.-S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Google Scholar) that expression of TRPV6 with substitution of FEL to AAA in the proposed pore region induced suppression of endogenous ICRAC in Jurkat cells. However, it is not clear whether the endogenous TRPV6 in Jurkat cells is required for ICRAC. Furthermore, TRPV6 displayed both store-operated and store-independent activities. The data we have presented above are significant because they demonstrate the following: (i) endogenous TRPC1 in HSG cells is required for the endogenous SOCE detected in these cells; (ii) expressed TRPC1 is regulated by store depletion and is not spontaneously active; and (iii) acidic aa residues in the pore region of TRPC1 contribute to SOCE.Although presently we do not have evidence for homo-multimerization of endogenous TRPC1 in HSG cells, we have shown that the expressed mutant TRPC1 is immunoprecipitated with endogenous TRPC1. This result has major implications because it suggests that TRPC1 monomers interact with each other to form SOCC. Our data are consistent with reports suggesting that Drosophila TRP and TRPC1β homomultimerize via N-terminal interactions (32Engelke M. Friedrich O. Budde P. Schafer C. Niemann U. Zitt C. Jungling E. Rocks O. Luckhoff A. Frey J. FEBS Lett. 2002; 17 (193–199): 523Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar). We suggest that endogenous or exogenously expressed TRPC1 monomers associate with each other to form functional SOCCs. Thus, when full-length TRPC1 is expressed channel activity is increased. When mutant TRPC1s are overexpressed relative to the endogenous protein, the probability of mutant TRPC1 monomers associating with each other or with endogenous TRPC1 monomers is relatively high. As a result, aberrant SOCCs are formed which have decreased permeability for calcium. In contrast, in cells transfected with antisense TRPC1 or expressing TRPC1Δ567–793, where either depletion of TRPC1 or the pore region is deleted, respectively, there is a reduction in the total number of functional channels rather than a change in the channel properties. However, other proteins, including other TRPCs, might be co-assembled with endogenous TRPC1 and required for functional SOCC in HSG cells. Studies using heterologous expression have shown that TRPC1 interacts with TRPC3 (33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar), TRPC6 (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar), and TRPC4 and TRPC5 (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). The latter study also showed that endogenous heteromers of either TRPC1 and TRPC4 or TRPC1 and TRPC5 were co-immunoprecipitated from rat brain. TRPC1 expression altered the currents generated when either TRPC5 or TRPC4 was expressed alone, and furthermore, these currents were not activated by store depletion. Although it is unclear why TRPC1 forms different types of channels in different cells, it is important to note that the characteristics of the channel formed by heteromeric TRPC1 channels (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) appear to be considerably different from those of ISOCmeasured in HSG cells. Further studies will be required to determine which TRPC proteins are endogenously expressed in HSG cells and interact with endogenous TRPC1.In conclusion, the data presented here suggest that TRPC1 is a component of the functional (pore-forming) unit of SOCC in HSG cells. However, these data do not rule out the possibility that SOCC might be a heteromer of TRPC1 with other TRPCs (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Google Scholar, 30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar, 35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) or with other as yet unknown protein(s). We have shown earlier that in HSG cells TRPC1, like the Drosophila TRP (14Montell C. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1)Google Scholar, 36Li H.S. Montell C. J. Cell Biol. 2000; 150: 1411-1422Google Scholar), is assembled in a supramolecular protein complex with key proteins involved in the Ca2+ signaling cascade that leads to SOCC activation (26Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2002; 275: 11934-11942Google Scholar). We suggest that SOCC activity in any cell type will depend not only on the proteins that constitute its pore-forming unit but also other regulatory proteins that might affect its function, assembly, or localization. It will be important to determine whether differences in the molecular composition of the channel per se, or its regulation, account for the large variation in the characteristics of SOCCs seen in different cell types. Previous studies (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 18Singh B.B. Liu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 36483-36486Google Scholar, 19Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 20Antoniotti S. Lovisolo D. Fiorio Pla A. Munaron L. FEBS Lett. 2002; 510: 189-195Google Scholar, 21Xu S.Z. Beech D.J. Circ. Res. 2001; 88: 84-87Google Scholar, 22Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Google Scholar, 23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) from our laboratory, and others, provide convincing evidence that TRPC1 has a critical role in SOCE. Based on the presently available data two possible roles can be assigned to TRPC1: (i) it is a regulatory subunit of SOCC that is required for its activation or assembly, and (ii) it is a component of the SOCC pore that directly contributes to SOCC-mediated Ca2+ influx. The data presented above suggest that TRPC1 is a functional component of an SOCC in HSG cells that contributes to Ca2+ influx in response to stimulation by either agonist or Tg. We have shown that the characteristics of SOCC activity generated by expression of TRPC1 in HSG cells are similar to the endogenous SOCC activity in these cells. Consistent with this, expression of TRPC1 in HSG cells induces an increase in the amplitude of the SOCC-mediated current, ISOC, without altering its characteristics (23Singh B.B. Liu X. Tang J. Zhu M.X. Ambudkar I.S. Mol. Cell. 2002; 9: 739-750Google Scholar) (Fig. 4). We have also shown here and previously (17Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar) that endogenous TRPC1 in HSG cells is essential for SOCC activity. Tg- or CCh-stimulated SOCC activity, ISOC, and Ca2+ influx, as well as levels of endogenous TRPC1 in HSG cells are decreased by 80% or more in cells transfected with antisense-TRPC1. More significant is our finding that acidic amino acid residues in the proposed pore region of TRPC1 contribute to SOCE. We have shown that expression of TRPC1 with simultaneous mutations (Asp → Asn and Glu → Gln) of seven acidic aa residues in the proposed pore region suppressed endogenous SOCC activity in more than 80% of the cells. In the few cells expressing this mutant where channel activity was detected, there was an apparent decrease in the Ca2+ conductance. Importantly, the amplitude of ISOC in these cells was decreased, and there was a left shift in the reversal potential, suggesting a decrease in the Ca2+ permeability of the channel. Interestingly, the Na+ current mediated by Mut-pore was similar to that seen in TRPC1-cells. Thus, mutations in the acidic aa residues in the pore region do not appear to decrease SOCC activity per se. We have also demonstrated that two of these seven acidic amino acid residues, Glu-576 and Asp-581, are involved in SOCC-mediated Ca2+ influx. Substitution of either residue, E576K or D581K, induced a suppression of Tg-stimulated SOCE and ISOC. Notably, Glu-576 and Glu-615 are conserved in TRPC1, TRPC4, and TRPC5, although Asp-581 is present only in TRPC1. Our data also rule out the possibility that a decrease in SOCC activity in cells expressing mutant TRPC1 proteins is due to differences in protein expression or altered localization. In aggregate, these data support the proposal that TRPC1 is a component of the functional (i.e. pore-forming) unit of SOCC, rather than an associated regulatory protein. Although it is possible that a single mutation in an extracellular domain of TRPC1 might alter its interaction with an as yet unknown SOCC protein and lead to a decrease in Ca2+influx via the channel, it is highly unlikely. Previous studies have shown that Asp → Asn substitution in the pore region of TRPV4 decreased Ca2+ influx, although Asp → Lys substitutions completely blocked channel function (29Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Droogmans G. Bindels R.J. J. Biol. Chem. 2001; 276: 1020-1025Google Scholar). Substitution of conserved hydrophobic residues (LFW) in the TRPC6 pore region eliminated TRPC6-generated channel activity in HEK293 cells (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar). However, neither of these channels appear to be involved in SOCE. While this manuscript was in preparation, it was reported (31Cui J. Bian J.-S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Google Scholar) that expression of TRPV6 with substitution of FEL to AAA in the proposed pore region induced suppression of endogenous ICRAC in Jurkat cells. However, it is not clear whether the endogenous TRPV6 in Jurkat cells is required for ICRAC. Furthermore, TRPV6 displayed both store-operated and store-independent activities. The data we have presented above are significant because they demonstrate the following: (i) endogenous TRPC1 in HSG cells is required for the endogenous SOCE detected in these cells; (ii) expressed TRPC1 is regulated by store depletion and is not spontaneously active; and (iii) acidic aa residues in the pore region of TRPC1 contribute to SOCE. Although presently we do not have evidence for homo-multimerization of endogenous TRPC1 in HSG cells, we have shown that the expressed mutant TRPC1 is immunoprecipitated with endogenous TRPC1. This result has major implications because it suggests that TRPC1 monomers interact with each other to form SOCC. Our data are consistent with reports suggesting that Drosophila TRP and TRPC1β homomultimerize via N-terminal interactions (32Engelke M. Friedrich O. Budde P. Schafer C. Niemann U. Zitt C. Jungling E. Rocks O. Luckhoff A. Frey J. FEBS Lett. 2002; 17 (193–199): 523Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar). We suggest that endogenous or exogenously expressed TRPC1 monomers associate with each other to form functional SOCCs. Thus, when full-length TRPC1 is expressed channel activity is increased. When mutant TRPC1s are overexpressed relative to the endogenous protein, the probability of mutant TRPC1 monomers associating with each other or with endogenous TRPC1 monomers is relatively high. As a result, aberrant SOCCs are formed which have decreased permeability for calcium. In contrast, in cells transfected with antisense TRPC1 or expressing TRPC1Δ567–793, where either depletion of TRPC1 or the pore region is deleted, respectively, there is a reduction in the total number of functional channels rather than a change in the channel properties. However, other proteins, including other TRPCs, might be co-assembled with endogenous TRPC1 and required for functional SOCC in HSG cells. Studies using heterologous expression have shown that TRPC1 interacts with TRPC3 (33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar), TRPC6 (30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar), and TRPC4 and TRPC5 (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). The latter study also showed that endogenous heteromers of either TRPC1 and TRPC4 or TRPC1 and TRPC5 were co-immunoprecipitated from rat brain. TRPC1 expression altered the currents generated when either TRPC5 or TRPC4 was expressed alone, and furthermore, these currents were not activated by store depletion. Although it is unclear why TRPC1 forms different types of channels in different cells, it is important to note that the characteristics of the channel formed by heteromeric TRPC1 channels (35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) appear to be considerably different from those of ISOCmeasured in HSG cells. Further studies will be required to determine which TRPC proteins are endogenously expressed in HSG cells and interact with endogenous TRPC1. In conclusion, the data presented here suggest that TRPC1 is a component of the functional (pore-forming) unit of SOCC in HSG cells. However, these data do not rule out the possibility that SOCC might be a heteromer of TRPC1 with other TRPCs (5Irvine R.F. FEBS Lett. 1990; 263: 5-9Google Scholar, 30Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar, 33Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 34Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-277805Google Scholar, 35Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) or with other as yet unknown protein(s). We have shown earlier that in HSG cells TRPC1, like the Drosophila TRP (14Montell C. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1)Google Scholar, 36Li H.S. Montell C. J. Cell Biol. 2000; 150: 1411-1422Google Scholar), is assembled in a supramolecular protein complex with key proteins involved in the Ca2+ signaling cascade that leads to SOCC activation (26Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2002; 275: 11934-11942Google Scholar). We suggest that SOCC activity in any cell type will depend not only on the proteins that constitute its pore-forming unit but also other regulatory proteins that might affect its function, assembly, or localization. It will be important to determine whether differences in the molecular composition of the channel per se, or its regulation, account for the large variation in the characteristics of SOCCs seen in different cell types. We are grateful to Drs. Klaus Groschner, Martha Nowycky, and Michael Zhu for helpful comments during the preparation of this manuscript. We are also grateful to Dr. Mitchel Villereal for kindly providing us with antisense constructs for TRPC1, TRPC3, and TRPC4 and Dr. Craig Montell for the TRPC1-FLAG construct.
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