Stromal Interaction Molecule Proteins Research Articles

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1

A drop of endoplasmic reticulum Ca(2+) concentration triggers its Ca(2+) ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca(2+) entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.

STIM proteins: dynamic calcium signal transducers

Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca(2+)) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca(2+) stored within the ER lumen. As ER Ca(2+) is released to generate primary Ca(2+) signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca(2+)-selective Orai channels to mediate finely controlled Ca(2+) signals and to homeostatically balance cellular Ca(2+). Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.

Regulation of lymphocyte function by ORAI and STIM proteins in infection and autoimmunity

Store-operated Ca(2+) entry (SOCE) in cells of the immune system is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels that are formed by ORAI1 and its homologues ORAI2 and ORAI3. They are activated by stromal interaction molecules (STIM) 1 and 2 in response to depletion of endoplasmic reticulum Ca(2+) stores. Loss-of-function mutations in the human ORAI1 and STIM1 genes abolish CRAC channel function and SOCE in a variety of non-excitable cells including lymphocytes and other immune cells, resulting in a unique clinical syndrome termed CRAC channelopathy. It is dominated by severe immunodeficiency and autoimmunity due to impaired SOCE and defects in the function of several lymphocyte subsets. These include CD8(+) T cells, CD4(+) effector and regulatory T cells, natural killer (NK) cells and B cells. This review provides a concise discussion of the role of CRAC channels in these lymphocyte populations and the regulation of adaptive immune responses to infection, in autoimmunity and inflammation.

CRACing the Cluster

Platelet-derived growth factor (PDGF) signaling is implicated in a wide range of diseases, and PDGF drives the pathological responses in many vascular disorders and fibrotic diseases such as atherosclerosis, restenosis, pulmonary arterial hypertension, and pulmonary fibrosis. In vascular smooth muscle cells (VSMC), PDGF is a potent mitogen which promotes proliferation and migration and contributes to pathological vascular remodeling. Recent studies have demonstrated a role for PDGF in stromal interaction molecule 1 (STIM1)/Orai1-mediated Ca2+ influx in VSMC.1 Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) is critical for many cellular processes. A rise in [Ca2+]cyt is a major trigger for VSMC proliferation and contraction. One of the major routes of Ca2+ entry into cells is through store-operated Ca2+ (SOC) channels. On depletion of Ca2+ from the stores (sarcoplasmic or endoplasmic reticulum, SR/ER), a Ca2+ deficiency signal is transmitted to the SOC channels in the plasma membrane via STIM1. This causes the SOC to open, allowing Ca2+ to flow into the cytosol, a process referred to as store-operated Ca2+ entry (SOCE). The cytosolic Ca2+ is then sequestered into the stores by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), thus replenishing the stores. Studies have demonstrated a role for Ca2+ release-activated Ca2+ (CRAC) channels in SOCE, with STIM1 and Orai1 as the major components.2 Article, see p 66 The majority of STIM1 is expressed in the SR/ER membrane, where it senses decreased Ca2+ concentration in the SR/ER when inositol 1,4,5-triphosphate (IP3)-mediated activation of IP3 receptor induces Ca2+ release. STIM1 then undergoes a conformational change allowing STIM1 to multimerize and translocate …

Induced Expression of STIM1 Sensitizes Intestinal Epithelial Cells to Apoptosis by Modulating Store-Operated Ca2+ Influx

IntroductionApoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca2+. Canonical transient receptor potential channel-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca2+ channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, stromal interaction molecule 1 (STIM1), is shown to act as a store Ca2+ sensor, and it can rapidly translocate to the plasma membrane where it directly interacts with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity. MethodsStudies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-α (TNF-α)/cycloheximide (CHX), and intracellular free Ca2+ concentration ([Ca2+]cyt) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants. ResultsStable STIM1-transfected IEC-6 cells (IEC-STIM1) showed increased STIM1 protein expression (~5 fold) and displayed a sustained increase in Ca2+ influx after Ca2+ store depletion (~2 fold). Susceptibility of IEC-STIM1 cells to TNF-α/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~20% in parental IEC-6 cells to ~40% in stable IEC-STIM1 cells 4 h after exposure to TNF-α/CHX (p < 0.05). In addition, stable IEC-TRPC1 cells also exhibited an increased sensitivity to TNF-α/CHX-induced apoptosis, which was prevented by STIM1 silencing through siSTIM1 transfection. STIM1 silencing by siSTIM1 also decreased Ca2+ influx after store depletion in cells overexpressing TRPC1. Levels of Ca2+ influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to Ca2+-free medium also blocked increased sensitivity to apoptosis. ConclusionsThese results indicate that (1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and (2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca2+ influx.

1114 Competitive Binding of Stim1 3′-Untranslated Region to RNA-Binding Protein HuR and MicroRNA-195 Regulates Its Expression and Epithelial Restitution

Stromal interaction molecule-1 (STIM1) acts as a store Ca2+ sensor in intestinal epithelial cells (IECs) and plays an important role in the restitution after injury, but the exact mechanism that regulates STIM1 abundance remains unknown. mRNA turnover and translation are major posttranscriptional processes of gene expression and are governed primarily by RNAbinding proteins (RBPs) and microRNAs (miRNAs). HuR is a RBP that associates with labile mRNAs bearing AU-rich elements and regulates the stability and translation of its target transcripts. Several studies show that RBPs and miRNAs can function jointly in the regulation of a shared target mRNA. Based on computationally predicted HuR and miRNA-195 (miR195) associations with the STIM1 mRNA, this study tests the hypothesis that HuR and miR195 jointly regulate STIM1 expression in IECs and therefore the epithelial restitution after injury. Methods: Studies were conducted in differentiated IECs (IEC-Cdx2L1 line) induced by stable Cdx2-transfection. The interaction of HuR and STIM1 mRNA was detected by biotin pull-down and RNP-IP assays, whereas miR-195 binding to the STIM1 transcript was examined by RNA pull-down assay using biotin-labeled miR-195. The stability of STIM1 mRNA was examined by measuring its half-life with real-time PCR analysis. HuR and miR-195 functions were investigated by siRNA silencing and ectopic gene overexpression. Epithelial restitution was measured by examining cell migration after wounding. Results: STIM1 mRNA is a target of HuR and miR-195, and both HuR and miR-195 directly bound to the STIM1 3'-UTR but not to its the coding region or 5'-UTR. Mapping experiments and studies using heterologous reporter constructs revealed that there were significant overlaps between HuRand miR-195-binding sites on the STIM1 3'-UTR. HuR silencing reduced HuR/STIM1 mRNA complex, increased the levels of STIM1 transcript associated with miR195, destabilized STIM1 mRNA (by ~50%), thus inhibiting STIM1 expression. Ectopic HuR overexpression increased its binding to the STIM1 mRNA, decreased the abundance of miR195/STIM1mRNA complex, rendered the STIM1mRNA stable, and increased STIM1 protein. In contrast, overexpression of a miR-195 precursor increased miR-195/STIM1 complex, reduced HuR association with STIM1 mRNA, and destabilized STIM1 mRNA, thereby decreasing STIM1 expression levels. miR-195 overexpression also inhibited epithelial restitution in an In Vitro model as indicated by decrease in cell migration after wounding, which was prevented by HuR overexpression. Conclusions: These results indicate that 1) HuR and miR-195 are novel regulators of STIM1 mRNA stability and 2) competitive binding of HuR and miR-195 to the STIM1 3'-UTR regulates STIM1 expression and intestinal epithelial restitution after injury.

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca 2+ oscillations and gene expression

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

Physiological and pathophysiological functions of SOCE in the immune system

Calcium signals play a critical role in many cell-type specific effector functions during innate and adaptive immune responses. The predominant mechanism to raise intracellular (Ca²⁺) used by most immune cells is store-operated Ca²⁺ entry (SOCE), whereby the depletion of endoplasmic reticulum (ER) Ca²⁺ stores triggers the influx of extracellular Ca²⁺. SOCE in immune cells is mediated by the highly Ca²⁺ selective Ca²⁺-release-activated Ca²⁺ (CRAC) channel, encoded by ORAI1, ORAI2 and ORAI3 genes. ORAI proteins are activated by stromal interaction molecules (STIM) 1 and 2, which act as sensors of ER Ca²⁺ store depletion. The importance of SOCE mediated by STIM and ORAI proteins for immune function is evident from the immunodeficiency and autoimmunity in patients with mutations in STIM1 and ORAI1 genes. These patients and studies in gene-targeted mice have revealed an essential role for ORAI/STIM proteins in the function of several immune cells. This review focuses on recent advances made towards understanding the role of SOCE in immune cells with an emphasis on the immune dysregulation that results from defects in SOCE in human patients and transgenic mice.

Physiological and pathophysiological functions of SOCE in the immune system

Calcium signals play a critical role in many cell-type specific effector functions during innate and adaptive immune responses. The predominant mechanism to raise intracellular (Ca²⁺) used by most immune cells is store-operated Ca²⁺ entry (SOCE), whereby the depletion of endoplasmic reticulum (ER) Ca²⁺ stores triggers the influx of extracellular Ca²⁺. SOCE in immune cells is mediated by the highly Ca²⁺ selective Ca²⁺-release-activated Ca²⁺ (CRAC) channel, encoded by ORAI1, ORAI2 and ORAI3 genes. ORAI proteins are activated by stromal interaction molecules (STIM) 1 and 2, which act as sensors of ER Ca²⁺ store depletion. The importance of SOCE mediated by STIM and ORAI proteins for immune function is evident from the immunodeficiency and autoimmunity in patients with mutations in STIM1 and ORAI1 genes. These patients and studies in gene-targeted mice have revealed an essential role for ORAI/STIM proteins in the function of several immune cells. This review focuses on recent advances made towards understanding the role of SOCE in immune cells with an emphasis on the immune dysregulation that results from defects in SOCE in human patients and transgenic mice.

STIM1-dependent store-operated Ca2+ entry is required for pathological cardiac hypertrophy

Alterations in intracellular Ca2+ homeostasis are an important trigger of pathological cardiac remodeling; however, mechanisms governing context-dependent changes in Ca2+ influx are poorly understood. Store-operated Ca2+ entry (SOCE) is a major mechanism regulating Ca2+ trafficking in numerous cell types, yet its prevalence in adult heart and possible role in physiology and disease are each unknown. The Ca2+-binding protein, stromal interaction molecule 1 (STIM1), is a Ca2+ sensor in the sarcoplasmic reticulum (SR), capable of triggering SOCE by interacting with plasma membrane Ca2+ channels. We report that SOCE is abundant and robust in neonatal cardiomyocytes; however, SOCE is absent from adult cardiomyocytes. Levels of STIM1 transcript and protein correlate with the amplitude of SOCE, and manipulation of STIM1 protein levels (via shRNA) or activity (via expression of constitutively active or dominant-negative mutants) reveals a critical role for STIM1 in activating SOCE in cardiac myocytes. In neonatal hearts a recently identified STIM1 splice variant (STIM1L) is predominant but diminishes with maturation, only to reemerge with agonist- or afterload-induced cardiac stress. To test for pathophysiological relevance, we evaluated both in vitro and in vivo models of cardiac hypertrophy, finding that STIM1 expression is re-activated by pathological stress to trigger significant SOCE-dependent Ca2+ influx. STIM1 amplifies agonist-induced hypertrophy via activation of the calcineurin–NFAT pathway. Importantly, inhibition of STIM1 suppresses agonist-triggered hypertrophy, pointing to a requirement for SOCE in this remodeling response. Stress-triggered STIM1 re-expression, and consequent SOCE activation, are critical elements in the upstream, Ca2+-dependent control of pathological cardiac hypertrophy.

Calcium entry via ORAI1 regulates glioblastoma cell proliferation and apoptosis

Introduction Calcium entry plays a critical role in the proliferation and survival of certain tumors. Ca 2+ release activated Ca 2+ (CRAC) channels constitute one of the most important pathways for calcium entry especially that of store-operated calcium entry (SOCE). ORAI1 and stromal interaction molecule1 (STIM1) are essential protein components of CRAC channels. In this study we tested the effect of inhibiting CRAC through ORAI1 and STIM1 on glioblastoma multiforme (GBM) tumor cell proliferation and survival. Methods Two glioblastoma cell lines, C6 (rat) and U251 (human), were used in the study. ORAI1 and STIM1 expressions were examined using Western blot and immunohistochemistry. CRAC channel activity and its components were inhibited with ion channel blockers and using siRNA knockdown. Changes in intracellular calcium concentration were recorded using Fura-2 fluorescent calcium imaging. Cell proliferation and apoptosis were examined using MTS and TUNEL assays, respectively. Results CRAC blockers, such as SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole), 2-aminoethoxydiphenyl borate (2-APB) and Diethylstilbestrol (DES), inhibited cell proliferations and SOCE in GBM cells. Knockdown of ORAI1 and STIM1 proteins using siRNA significantly inhibited C6 cell proliferation and SOCE compared with those in control cells, and a more significant effect was observed in cells with ORAI1 siRNA knockdown than that of STIM1-treated cells. Both CRAC blockers and siRNA treatments increased apoptosis in C-6 cells compared with control. Conclusion Calcium entry via ORAI1 and CRAC channels are important for GBM proliferation and survival.

STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

Store‐operated calcium entry remains fully functional in aged mouse skeletal muscle despite a decline in STIM1 protein expression

Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundant STIM1 and Orai1 in the junctional membranes. The precise role of SOCE in skeletal muscle Ca(2+) homeostasis and excitation-contraction coupling remains to be defined. Regardless, it remains important to determine whether the function and capacity of SOCE changes in aged skeletal muscle. We identified an approximate 40% decline in the expression of the integral SOCE protein, stromal interacting molecule 1 (STIM1), but no such decline in its coupling partner, Orai1, in muscle fibers from aged mice. To determine whether this changed aspects of SOCE functionality in skeletal muscle in aged mice, Ca(2+) in the cytoplasm and t-system were continuously and simultaneously imaged on a confocal microscope during sarcoplasmic reticulum Ca(2+) release and compared to experiments under identical conditions using muscle fibers from young mice. Normal activation, deactivation, Ca(2+) influx, and spatiotemporal characteristics of SOCE were found to persist in skeletal muscle from aged mice. Thus, SOCE remains a robust mechanism in aged skeletal muscle despite the decline in STIM1 protein expression, suggesting STIM1 is in excess in young skeletal muscle.

Auto-inhibitory role of the EF-SAM domain of STIM proteins in store-operated calcium entry

Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca(2+)) sensors that differentially regulate plasma membrane Ca(2+) release activated Ca(2+) channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca(2+)-binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing "super-unstable" or "super-stable" STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca(2+)- and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca(2+) entry by STIM1 and STIM2.

Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca(2+) entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid

The mechanism behind Ca(2)(+) entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca(2)(+) channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca(2)(+) caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca(2)(+) entry (SOCE). Candidate molecules for non-voltage-operated Ca(2)(+) signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca(2)(+) release-activated Ca(2)(+) modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.

CRAC channelopathies

Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway in many non-excitable and some excitable cells. It is regulated by the filling state of intracellular Ca2+ stores, notably the endoplasmic reticulum (ER). Reduction in [Ca2+]ER results in activation of plasma membrane Ca2+ channels that mediate sustained Ca2+ influx which is required for many cell functions as well as refilling of Ca2+ stores. The Ca2+ release activated Ca2+ (CRAC) channel is the best characterized SOC channel with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel, long mysterious, have been defined. ORAI1 (or CRACM1) acts as the pore-forming subunit of the CRAC channel in the plasma membrane. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca2+]ER, and activates the CRAC channel upon store depletion by binding to ORAI1. Both proteins are widely expressed in many tissues in both human and mouse consistent with the widespread prevalence of SOCE and CRAC channel currents in many cells types. CRAC channelopathies in human patients with mutations in STIM1 and ORAI1 are characterized by abolished CRAC channel currents, lack of SOCE and-clinically-immunodeficiency, congenital myopathy, and anhydrotic ectodermal dysplasia. This article reviews the role of ORAI and STIM proteins for SOCE and CRAC channel function in a variety of cell types and tissues and compares the phenotypes of ORAI1 and STIM1-deficient human patients and mice with targeted deletion of Orai and Stim genes.

STIM1 gates the store-operated calcium channel ORAI1 in vitro

Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.

Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.