Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

The ER Ca2+ sensor STIM1 and the Ca2+ channel Orai1 are key players in store-operated Ca2+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca2+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca2+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca2+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca2+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca2+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca2+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca2+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.     

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.     

Competitive modulation of Ca2+ release-activated Ca2+ channel gating by STIM1 and 2-aminoethyldiphenyl borate

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.     

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.     

Capacitative and non-capacitative signaling complexes in human platelets

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.     

Molecular Determinants Mediating Gating of Transient Receptor Potential Canonical (TRPC) Channels by Stromal Interaction Molecule 1 (STIM1)

Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca²⁺ influx. TRPCs are gated open by the endoplasmic reticulum Ca²⁺ sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca²⁺ influx, and inhibition of Trpc3 had no further effect on Ca²⁺ influx in Trpc1^−/− cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.     

Molecular determinants within N terminus of Orai3 protein that control channel activation and gating

STIM1 and Orai represent the key components of Ca(2+) release-activated Ca(2+) channels. Activation of Orai channels requires coupling of the C terminus of STIM1 to the N and C termini of Orai. Although the latter appears to be central in the interaction with STIM1, the role of the N terminus and particularly of the conserved region close to the first transmembrane sequence is less well understood. Here, we investigated in detail the functional role of this conserved region in Orai3 by stepwise deletions. Molecular determinants were mapped for the two modes of Orai3 activation via STIM1 or 2-aminoethoxydiphenyl borate (2-APB) and for current gating characteristics. Increasing N-terminal truncations revealed a progressive decrease of the specific fast inactivation of Orai3 concomitant with diminished binding to calmodulin. STIM1-dependent activation of Orai3 was maintained as long as the second half of this conserved N-terminal domain was present. Further truncations abolished it, whereas Orai3 stimulation via 2-APB was partially retained. In aggregate, the N-terminal conserved region plays a multifaceted role in Orai3 current gating with distinct structural requirements for STIM1- and 2-APB-stimulated activation.     

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.     

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.     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells: IMPORTANCE OF PROTEIN KINASE C δ (PKCδ) AND STROMAL INTERACTION MOLECULE 2 (STIM2)*

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca²⁺-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca²⁺-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.     

Store-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes

Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca(2+) entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) in neutrophil-like HL-60 cells. When extracellular Ca(2+) is initially removed, early addition of Ca(2+) after stimulation causes a complete restoration of Ca(2+) entry and H(2)O(2) production. Both Ca(2+) entry and H(2)O(2) production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca(2+) channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca(2+) influx and H(2)O(2) production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca(2+) entry and H(2)O(2) production observed in cells transfected by TRPC3 siRNA. In summary, Ca(2+) influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE.     

Impaired Orai1-mediated resting Ca2+ entry reduces the cytosolic [Ca2+] and sarcoplasmic reticulum Ca2+ loading in quiescent junctophilin 1 knock-out myotubes

In the absence of store depletion, plasmalemmal Ca(2+) permeability in resting muscle is very low, and its contribution in the maintenance of Ca(2+) homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca(2+) entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca(2+) entry pathway on overall Ca(2+) homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca(2+) entry, [Ca(2+)](rest), and intracellular Ca(2+) content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca(2+) entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca(2+) homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca(2+) homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca(2+) permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca(2+)](rest) and resting Ca(2+) stores and that this pathway is defective in JP1 KO myotubes.     

Skeletal muscle dressed in SOCs

Store-operated Ca(2+) channels (SOCs) are activated in response to Ca(2+) release from the endoplasmic reticulum (ER). The stromal interaction molecule 1 (STIM1) is the ER sensor that transmits the stored Ca(2+) content to the pore-forming SOCs Orai and TRPC channels. Recent studies reveal high levels of Orai1 and STIM1 in skeletal muscle, and a prominent role of SOCs in muscle development and function.     

Temperature-dependent STIM1 activation induces Ca²+ influx and modulates gene expression

Intracellular Ca(2+) is essential for diverse cellular functions. Ca(2+) entry into many cell types including immune cells is triggered by depleting endoplasmic reticulum (ER) Ca(2+), a process termed store-operated Ca(2+) entry (SOCE). STIM1 is an ER Ca(2+) sensor. Upon Ca(2+) store depletion, STIM1 clusters at ER-plasma membrane junctions where it interacts with and gates Ca(2+)-permeable Orai1 ion channels. Here we show that STIM1 is also activated by temperature. Heating cells caused clustering of STIM1 at temperatures above 35 °C without depleting Ca(2+) stores and led to Orai1-mediated Ca(2+) influx as a heat off-response (response after cooling). Notably, the functional coupling of STIM1 and Orai1 is prevented at high temperatures, potentially explaining the heat off-response. Additionally, physiologically relevant temperature shifts modulate STIM1-dependent gene expression in Jurkat T cells. Therefore, temperature is an important regulator of STIM1 function.     

cAMP induces stromal interaction molecule 1 (STIM1) puncta but neither Orai1 protein clustering nor store-operated Ca2+ entry (SOCE) in islet cells

The events leading to the activation of store-operated Ca(2+) entry (SOCE) involve Ca(2+) depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca(2+) channel protein Orai1 to activate Ca(2+) influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing β-cells and glucagon-secreting α-cells within intact mouse and human pancreatic islets. ER Ca(2+) depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca(2+) store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in α- than in β-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in α-cells and retranslocation in β-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.     

源自天蚕素A和爪蟾素2的杂合肽P18体外抑制内皮细胞血管生成

目的探讨杂合肽P18体外对内皮细胞EA.hy926血管生成的抑制作用.方法采用MTT法检测P18对EA.hy926细胞增殖的影响;应用Matrigel实验检测P18对内皮细胞形成管状结构的影响;利用流式细胞术分析P18对内皮细胞的损伤作用.结果 MTT结果显示P18可明显抑制EA.hy926细胞的增殖,且抑制率存在剂量依赖性;Matrigel实验表明P18具有抑制EA.hy926细胞体外分化成管状结构的作用;流式结果显示15 μM P18作用内皮细胞6 h后,所诱导的细胞坏死比例达到81.4%.结论体外实验结果表明,杂合肽P18具有体外抑制EA.hy926细胞血管生成的作用.     

Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.     

TRPC channels as STIM1-regulated store-operated channels

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 demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.