A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

CRAC channels and disease – From human CRAC channelopathies and animal models to novel drugs

Ca²⁺ release-activated Ca²⁺ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca²⁺ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders.     

Role of STIM1/ORAI1-mediated store-operated Ca2+ entry in skeletal muscle physiology and disease

Store-operated Ca²⁺ entry (SOCE) is a Ca²⁺ entry mechanism activated by depletion of intracellular Ca²⁺ stores. In skeletal muscle, SOCE is mediated by an interaction between stromal-interacting molecule-1 (STIM1), the Ca²⁺ sensor of the sarcoplasmic reticulum, and ORAI1, the Ca²⁺-release-activated-Ca²⁺ (CRAC) channel located in the transverse tubule membrane. This review focuses on the molecular mechanisms and physiological role of SOCE in skeletal muscle, as well as how alterations in STIM1/ORAI1-mediated SOCE contribute to muscle disease. Recent evidence indicates that SOCE plays an important role in both muscle development/growth and fatigue. The importance of SOCE in muscle is further underscored by the discovery that loss- and gain-of-function mutations in STIM1 and ORAI1 result in an eclectic array of disorders with clinical myopathy as central defining component. Despite differences in clinical phenotype, all STIM1/ORAI1 gain-of-function mutations-linked myopathies are characterized by the abnormal accumulation of intracellular membranes, known as tubular aggregates. Finally, dysfunctional STIM1/ORAI1-mediated SOCE also contributes to the pathogenesis of muscular dystrophy, malignant hyperthermia, and sarcopenia. The picture to emerge is that tight regulation of STIM1/ORAI1-dependent Ca²⁺ signaling is critical for optimal skeletal muscle development/function such that either aberrant increases or decreases in SOCE activity result in muscle dysfunction.     

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca²⁺ release‐activated Ca²⁺ (CRAC) current having an important role in T‐cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca²⁺ stores are depleted. Here we show, utilizing a novel STIM1‐derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C‐terminal, intramolecular transition into an extended conformation when activating ORAI1. The C‐terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled‐coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full‐length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.     

CRAC channels in dental enamel cells

Enamel mineralization relies on Ca²⁺ availability provided by Ca²⁺ release activated Ca²⁺ (CRAC) channels. CRAC channels are modulated by the endoplasmic reticulum Ca²⁺ sensor STIM1 which gates the pore subunit of the channel known as ORAI1, found the in plasma membrane, to enable sustained Ca²⁺ influx. Mutations in the STIM1 and ORAI1 genes result in CRAC channelopathy, an ensemble of diseases including immunodeficiency, muscular hypotonia, ectodermal dysplasia with defects in sweat gland function and abnormal enamel mineralization similar to amelogenesis imperfecta (AI). In some reports, the chief medical complain has been the patient’s dental health, highlighting the direct and important link between CRAC channels and enamel. The reported enamel defects are apparent in both the deciduous and in permanent teeth and often require extensive dental treatment to provide the patient with a functional dentition. Among the dental phenotypes observed in the patients, discoloration, increased wear, hypoplasias (thinning of enamel) and chipping has been reported. These findings are not universal in all patients. Here we review the mutations in STIM1 and ORAI1 causing AI-like phenotype, and evaluate the enamel defects in CRAC channel deficient mice. We also provide a brief overview of the role of CRAC channels in other mineralizing systems such as dentine and bone.     

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.     

Role of the STIM1 C-terminal Domain in STIM1 Clustering

Store-operated Ca²⁺ entry (SOCE) represents a ubiquitous Ca²⁺ influx pathway activated by the filling state of intracellular Ca²⁺ stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca²⁺ sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca²⁺ stores are full, these domains are inactive to prevent constitutive Ca²⁺ entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca²⁺ stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.     

The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

Ca^2 + elevation is essential to platelet activation. STIM1 senses Ca^2 + in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca^2 + entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca^2 + entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca^2 + entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca^2 +-sensing role of STIM1 is served by the protein in the ER.     

No social distancing between ORAI channels

ORAI1 is established as an essential component of Ca²⁺ release-activated Ca²⁺ (CRAC) channel which mediates store-operated Ca²⁺ entry (SOCE). However, the contributions of ORAI2 and ORAI3 to SOCE are not understood. We highlight a recent study which shows that ORAI proteins form heteromeric channels which tune SOCE over a range of stimulus intensities.     

STIM1 Is a Novel Component of ER-Chlamydia trachomatis Inclusion Membrane Contact Sites

Productive developmental cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis depends on the interaction of the replicative vacuole, named the inclusion, with cellular organelles. We have recently reported the formation of ER-Inclusion membrane contact sites (MCSs), where the endoplasmic reticulum (ER) is in apposition to the inclusion membrane. These platforms contain the C. trachomatis inclusion membrane protein IncD, the mammalian ceramide transfer protein CERT and the ER resident proteins VAPA/B and were proposed to play a role in the non-vesicular trafficking of lipids to the inclusion. Here, we identify STIM1 as a novel component of ER-Inclusion MCSs. STIM1, an ER calcium (Ca²⁺) sensor that relocate to ER-Plasma Membrane (PM) MCSs upon Ca²⁺ store depletion, associated with C. trachomatis inclusion. STIM1, but not the general ER markers Rtn3C and Sec61ß, was enriched at the inclusion membrane. Ultra-structural studies demonstrated that STIM1 localized to ER-Inclusion MCSs. Time-course experiments showed that STIM1, CERT and VAPB co-localized throughout the developmental cycle. By contrast, Orai1, the PM Ca²⁺ channel that interacts with STIM1 at ER-PM MCSs, did not associate with C. trachomatis inclusion. Upon ER Ca²⁺ store depletion, a pool of STIM1 relocated to ER-PM MCSs, while the existing ER-Inclusion MCSs remained enriched in STIM1. Finally, we have identified the CAD domain, which mediates STIM1-Orai1 interaction, as the minimal domain required for STIM1 enrichment at ER-Inclusion MCSs. Altogether this study identifies STIM1 as a novel component of ER-C. trachomatis inclusion MCSs. We discuss the potential role(s) of STIM1 during the infection process.     

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle

Skeletal muscle fibres support store-operated Ca²⁺-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca²⁺]_SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca²⁺-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction.Here we show that phasic SOCE is not only shaped by global [Ca²⁺]_SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca²⁺]_SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca²⁺ release. We derive a delay of 0.3 ms reflecting diffusive Ca²⁺-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca²⁺-release. Numerical simulations of Ca²⁺-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca²⁺-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca²⁺-STIM1 binding suggests a Ca²⁺ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only μs delays, because [Ca²⁺] in the terminal cisternae is just above the threshold for Ca²⁺ dissociation from STIM1 under physiological resting conditions.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

We have investigated the molecular basis of intracellular Ca²⁺ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca²⁺ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca²⁺ levels, agonist-induced Ca²⁺ increases, store-operated Ca²⁺ entry (SOCE), and store-operated currents (I_SOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca²⁺ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I_CRAC plus a nonselective I_SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca²⁺ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca²⁺ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca²⁺ remodeling and tumor features in colon cancer.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.     

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-β-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.     

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca²⁺ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca²⁺ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na⁺ that is critical in activating the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) causing enhanced mitochondrial Na⁺ uptake and Ca²⁺ efflux. Omission of extracellular Na⁺ prevents the cytosolic Na⁺ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na⁺ and Ca²⁺ signals to effect mitochondrial redox control over SOCE.