Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components

Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca2+ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca2+-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I(SOC) in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I(SOC) in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca2+ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.     

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (Ca2+L) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca2+L channels and lead to Ca2+ entry and vasoconstriction.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins

Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca(2+) signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca(2+) signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca(2+) oscillations via IP(3) -mediated depletion of intracellular stores and activation of store-operated Ca(2+) (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca(2+) add-back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca(2+) oscillations were comparatively short-lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short-lived oscillatory response into a long-lived one. Thus, we conclude that SLO-mediated Ca(2+) signalling involves Ca(2+) release from intracellular stores and STIM1/Orai1-dependent SOC entry. The phenotype of Ca(2+) signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry-associated proteins in S. pyogenes-induced lung infection and pneumonia.     

TRPC channels as STIM1-regulated SOCs

Store-operated Ca2+ channels (SOCs) are Ca2+ influx channels at the plasma membrane whose opening is determined by the level of Ca2+ stored in the endoplasmic reticulum lumen. The SOCs are activated in response to receptor-mediated or passive depletion of ER Ca2+ to regulate many Ca2+-dependent cellular functions. Early work implicated the TRPC channels as SOCs. More recently, it was found that the Orai channels mediate the CRAC current and that the Ca2+ binding protein STIM1 functions as the ER Ca2+ sensor that mediates activation of the SOCs in response to depletion of ER Ca2+. Key questions are whether both TRPC channels and the Orais are opened by STIM1 and the molecular mechanism by which STIM1 opens the SOCs. Ample biochemical and functional evidence indicate interaction of the TRPC channels with STIM1. Furthermore, it was found that STIM1 gates TRPC channels by electrostatic interaction of STIM1(K684,K685) in the polybasic domain of STIM1 with two negative charges (aspartates or glutamates) that are conserved in all TRPC channels. Charge mutants of STIM1(K684,K685) and TRPC1(D639,D640) and TRPC3(D697D698) were used to develop further direct evidence for the function of TRPC channels as SOCs. The evidence in favor of TRPC channels as SOCs are discussed.     

Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1

Store-operated Ca2+ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca2+ stores that requires the participation of the Ca2+ sensor STIM1, which communicates the Ca2+ content of the stores to the plasma membrane Ca2+-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca2+ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-beta-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca2+ stores and attenuates thapsigargin-evoked Ca2+ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca2+ stores.     

A novel mechanism for the store-operated calcium influx pathway

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.     

TRPC1: the link between functionally distinct store-operated calcium channels

Although store-operated calcium entry (SOCE) was identified more that two decades ago, understanding the molecular mechanisms that regulate and mediate this process continue to pose a major challenge to investigators in this field. Thus, there has been major focus on determining which of the models proposed for this mechanism is valid and conclusively establishing the components of the store-operated calcium (SOC) channel(s). The transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the elusive store-operated Ca(2+) entry channel. While all TRPCs are activated in response to agonist-stimulated phosphatidylinositol 4,5, bisphosphate (PIP(2)) hydrolysis, only some display store-dependent regulation. TRPC1 is currently the strongest candidate component of SOC and is shown to contribute to SOCE in many cell types. Heteromeric interactions of TRPC1 with other TRPCs generate diverse SOC channels. Recent studies have revealed novel components of SOCE, namely the stromal interacting molecule (STIM) and Orai proteins. While STIM1 has been suggested to be the ER-Ca(2+) sensor protein relaying the signal to the plasma membrane for activation of SOCE, Orai1 is reported to be the pore-forming component of CRAC channel that mediates SOCE in T-lymphocytes and other hematopoetic cells. Several studies now demonstrate that TRPC1 also associates with STIM1 suggesting that SOC and CRAC channels are regulated by similar molecular components. Interestingly, TRPC1 is also associated with Orai1 and a TRPC1-Orai1-STIM1 ternary complex contributes to SOC channel function. This review will focus on the diverse SOC channels formed by TRPC1 and the suggestion that TRPC1 might serve as a molecular link that determines their regulation by store-depletion.     

Sphingosine 1-phosphate-induced mobilization of intracellular Ca2+ mediates rac activation and adherens junction assembly in endothelial cells

Sphingosine 1-phosphate (S1P) ligation of endothelial differentiation gene-1 receptor coupled to the heterotrimeric G protein, Gi, promotes endothelial barrier strengthening via Rac-dependent assembly of adherens junctions (AJs). However, the mechanism of Rac activation induced by S1P stimulation remains unclear. In live endothelial cells expressing GFP-Rac, we observed that S1P induced the translocation of Rac to intercellular junctions, resulting in junctional sealing. We investigated the role of intracellular Ca2+ in signaling Rac activation and the enhancement of endothelial barrier function. We observed that S1P activated the release of Ca2+ from endoplasmic reticulum stores, and subsequent Ca2+ entry via lanthanum-sensitive store-operated Ca2+ channels (SOC) after store depletion. Inhibition of Gi, phospholipase C, or inositol trisphosphate receptor prevented the S1P-activated increase in intracellular Ca2+ as well as Rac activation, AJ assembly, and enhancement of endothelial barrier. Chelation of intracellular Ca2+ with BAPTA blocked S1P-induced Rac activation, indicating the requirement for Ca2+ in the response. Inhibition of SOC by lanthanum or transient receptor potential channel 1 (TRPC1), a SOC constituent, by TRPC1 antibody, failed to prevent S1P-induced Rac translocation to junctions and AJ assembly. Thus, our results demonstrate that S1P promotes endothelial junctional integrity by activating the release of endoplasmic reticulum-Ca2+, which induces Rac activation and promotes AJ annealing.     

Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 microM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 microM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.     

RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+]i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability.     

Remodelling of the endoplasmic reticulum during store-operated calcium entry

SOCE (store-operated calcium entry) is a ubiquitous cellular mechanism linking the calcium depletion of the ER (endoplasmic reticulum) to the activation of PM (plasma membrane) Ca2+-permeable channels. The activation of SOCE channels favours the entry of extracellular Ca2+ into the cytosol, thereby promoting the refilling of the depleted ER Ca2+ stores as well as the generation of long-lasting calcium signals. The molecules that govern SOCE activation comprise ER Ca2+ sensors [STIM1 (stromal interaction molecule 1) and STIM2], PM Ca2+-permeable channels {Orai and TRPC [TRP (transient receptor potential) canonical]} and regulatory Ca2+-sensitive cytosolic proteins {CRACR2 [CRAC (Ca2+ release-activated Ca2+ current) regulator 2]}. Upon Ca2+ depletion of the ER, STIM molecules move towards the PM to bind and activate Orai or TRPC channels, initiating calcium entry and store refilling. This molecular rearrangement is accompanied by the formation of specialized compartments derived from the ER, the pre-cER (cortical ER) and cER. The pre-cER appears on the electron microscope as thin ER tubules enriched in STIM1 that extend along microtubules and that are devoid of contacts with the PM. The cER is located in immediate proximity to the PM and comprises thinner sections enriched in STIM1 and devoid of chaperones that might be dedicated to calcium signalling. Here, we review the molecular interactions and the morphological changes in ER structure that occur during the SOCE process.     

STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

Biochemical and functional properties of the store-operated Ca2+ channels

Store-operated calcium entry (SOCE) is a major mechanism for Ca²⁺ entry in excitable and non-excitable cells. The best-characterised store-operated current is I_CRAC, but other currents activated by Ca²⁺ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca²⁺ sensor that communicates the content of the Ca²⁺ stores to the store-operated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and co-clustering with the Ca²⁺ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function.     

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells

Store-operated Ca²⁺ channels (SOCs) are voltage-independent Ca²⁺ channels activated upon depletion of the endoplasmic reticulum Ca²⁺ stores. Early studies suggest the contribution of such channels to Ca²⁺ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca²⁺ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca²⁺ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca²⁺ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.     

Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.