Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

Receptor-activated Ca 2+ influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca 2+ influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca 2+ influx channels. The findings that STIM1 is an ER resident Ca 2+ binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca 2+ stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels should further clarify their regulation by STIM1 and function as SOCs.

Ca2ϩ influx is a critical component of the receptor-evoked Ca 2ϩ signal and plays a role in many physiological functions (1). The best described form of Ca 2ϩ influx is mediated by the store-operated Ca 2ϩ channels (SOCs), 3 which are activated by agonist-dependent or agonist-independent depletion of Ca 2ϩ stored in the ER (1). The molecular identity of the SOCs and I crac is still not known with certainty, although recent work points to ORAI1/CRACM1/olf186-F as a potential I crac (2-5). However, accumulating evidence indicates that members of the transient receptor potential (TRP) family of ion channels are associated with SOCs in mammalian cells. Thus, deletion of TRPC4 in mice (6, 7) or of TRPC1, TRPC3, TRPC6, and TRPC7 by antisense or siRNA (8 -10) and dominant negative TRPC1, TRPC3, partially inhibit SOCs and/or receptor-stimulated Ca 2ϩ influx. The mechanism by which agonist stimulation activates Ca 2ϩ influx by TRPC channels is not well understood. TRPC1, -4, and -5 can be activated by store depletion, whereas TRPC3, -6, and -7 can be activated by the lipid diacylglycerol (11,15,16). However, depending on cell type and expression levels, TRPC3 can also be activated by store depletion (17)(18)(19). Several mechanisms have been proposed to explain how store depletion leads to activation of SOCs and TRPC channels; conformational coupling between TRPC channels and IP 3 receptors (IP 3 Rs) (18, 20 -22), exocytotic insertion of the channels in the plasma membrane (PM) (23)(24)(25), and activation by a diffusible messenger (26,27). Biochemical and functional evidence showed regulatory interaction between IP 3 Rs and several TRPC channels, including TRPC1 and TRPC3 (18,[28][29][30][31][32] Rs (30,32).A newly discovered and apparently a general regulatory mechanism of TRPC channel activity is agonist-stimulated translocation of the channels to the PM. In HEK293 cells, receptor stimulation but not passive store depletion was reported to stimulate the translocation of TRPC3 to the PM in a mechanism that was inhibited by cleavage of VAMP2 (vesicleassociated membrane protein 2) with tetanus toxin (38). Another form of regulation of TRPC3 is by interaction with phospholipase C␥ (22, 39). However, unlike the role of VAMP2, phospholipase C␥ does not affect the acute expression or translocation of TRPC3 but rather the steady-state level of TRPC3 in the PM (22). Stimulation of the epidermal growth factor receptor resulted in translocation of TRPC5 to the PM in a mechanism that was dependent on phosphoinositide 3-kinase, the Rho GTPase Rac1, and phosphatidylinositol-4-phosphate 5-kinase (PIP(5)K␣) (24). Finally, stimulation of the muscarinic M3 receptor resulted in translocation of TRPC6 to the PM in a time course that coincides with activation of Ca 2ϩ influx (25). For the most part, TRPC channel translocation has been studied in cell lines. Whether such a mechanism also operates in native cells is not known. Furthermore, TRPC3, -5, and -6 bind Homers and IP 3 Rs (33) (present work). The potential role of Homer and IP 3 R...

3 ؊ transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO 3 ؊ -transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO 3 ؊ transport by regulating the activity of all luminal HCO 3 ؊ secretion and salvage mechanisms of secretory epithelial cells. HCO 3Ϫ concentration is tightly controlled in all biological fluids including fluids secreted by exocrine glands. The ductal systems or their equivalents are the sites of active regulation of HCO 3 Ϫ content of the secreted fluids. This is also the site of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) 1 (1-5). The transporters participating in ductal HCO 3 Ϫ homeostasis and their regulation are only partially known. Probably, the best results are available in the salivary glands and pancreatic ducts. Active regulation of luminal HCO 3 Ϫ concentration and pH i requires the regulation of both HCO 3 Ϫ secretory and absorptive mechanisms. HCO 3 Ϫ secretion is believed to occur by HCO 3 Ϫ influx across the basolateral membrane mediated by a Na ϩ -HCO 3 Ϫ cotransport mechanism (6, 7). The transporter mediating this activity is probably pNBC1, the pancreatic isoform of the electrogenic Na ϩ -HCO 3 Ϫ cotransporter family (8, 9). HCO 3 Ϫ efflux across the luminal membrane (LM) requires the activity of a Cl Ϫ /HCO 3 Ϫ exchange mechanism (6, 10, 11) and is dependent on the expression of CFTR both in human and in animal models (11,12).In the resting state, secretory glands have to absorb HCO 3 Ϫ . The transporters involved in HCO 3 Ϫ absorption are only beginning to emerge. HCO 3 Ϫ influx across the LM is in part the result of Na ϩ /H ϩ exchange mediated by NHE3 (13,14). However, in recent studies with the pancreatic (13) and the submandibular gland (SMG) ducts (9), we showed that Ͼ50% HCO 3 Ϫ absorption (H ϩ secretion) is mediated by more than one Na ϩ -dependent mechanism that is different from any known NHE isoform. Furthermore, we found that the SMG duct and acinar cells express several splice variants of NBC3 (rat orthologues NBCn1B-D) and used anti-NBC3 antibodies to localize the proteins to the LM (...