STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca²⁺ sensor stromal interacting molecule 1 (STIM1) and the Ca²⁺ channel Orai1 mediate the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway activated by depletion of internal Ca²⁺ stores and mediated through the highly Ca²⁺-selective, Ca²⁺ release-activated Ca²⁺ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca²⁺ currents regulated by either arachidonate or its metabolite, leukotriene C₄. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca²⁺ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.     

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. Ca²⁺ release activated Ca²⁺ (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.     

Orai1 controls C5a‐induced neutrophil recruitment in inflammation

Stromal interaction molecule 1 (STIM1)‐dependent store operated calcium‐entry (SOCE) through Orai1‐mediated calcium (Ca²⁺) influx is considered a major pathway of Ca²⁺ signaling, serving T‐cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1‐deficient neutrophils present defects in fMLP and complement C5a‐induced Ca²⁺ influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1‐deficient neutrophils exhibit normal STIM1‐dependent SOCE and STIM1‐deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1^?/? chimeric mice show impaired neutrophil recruitment in LPS‐induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a‐triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1^?/? chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG‐induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1‐independent pathway of Ca²⁺‐influx in neutrophils.     

Orai1 Expression Is Closely Related with Favorable Prognostic Factors in Clear Cell Renal Cell Carcinoma

Store-operated calcium (Ca²⁺) entry (SOCE) is the principal Ca²⁺ entry route in non-excitable cells, including cancer cells. We previously demonstrated that Orai1 and STIM1, the molecular components of SOCE, are involved in tumorigenesis of clear cell renal cell carcinoma (CCRCC). However, a clinical relevance of Orai1 and STIM1 expression in CCRCC has been ill-defined. Here, we investigated the expression of Orai1 and STIM1 in CCRCC, and compared their expression with clinico-pathological parameters of CCRCC and the patients’ outcome. Immunohistochemical staining for Orai1 and STIM1 was performed on 126 formalin fixed paraffin embedded tissue of CCRCC and western blot analysis for Orai1 was performed on the available fresh tissue. The results were compared with generally well-established clinicopathologic prognostic factors in CCRCC and patient survival. Membrane protein Orai1 is expressed in the nuclei in CCRCC, whereas STIM1 shows the cytosolic expression pattern in immunohistochemical staining. Orai1 expression level is inversely correlated with CCRCC tumor grade, whereas STIM1 expression level is not associated with tumor grade. The higher Orai1 expression is significantly associated with lower Fuhrman nuclear grade, pathologic T stage, and TNM stage and with favorable prognosis. The expression level of STIM1 is not correlated with CCRCC grade and clinical outcomes. Orai1 expression in CCRCC is associated with tumor progression and with favorable prognostic factors. These results suggest that Orai1 is an attractive prognostic marker and therapeutic target for CCRCC.     

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.     

Functional transient receptor potential canonical type 1 channels in human atrial myocytes

Transient receptor potential (TRP) channels are not well understood in human atrium, and the present study was therefore designed to investigate whether TRPC channels would mediate the nonselective cation current reported previously and are involved in the formation of store-operated Ca²⁺ entry (SOCE) channels in human atrial myocytes using approaches of whole-cell patch voltage-clamp, RT-PCR, Western blotting, co-immunoprecipitation, and confocal scanning approaches, etc. We found that a nonselective cation current was recorded under K⁺-free conditions in human atrial myocytes, and the current was inhibited by the TRP channel blocker La³⁺. Thapsigargin enhanced the current, and its effect was suppressed by La³⁺ and prevented by pipette inclusion of anti-TRPC1 antibody. Endothlin-1 and angiotensin II enhanced the current that could be inhibited by La³⁺. Gene and protein expression of TRPC1 channels were abundant in human atria. In addition, mRNA and protein of STIM1 and Orai1, components of SOCE channels, were abundantly expressed in human atria. Co-immunoprecipitation analysis demonstrated an interaction of TRPC1 with STIM1 and/or Orai1. Ca²⁺ signaling mediated by SOCE channels was detected by a confocal microscopy technique. These results demonstrate the novel evidence that TRPC1 channels not only mediate the nonselective cation current, but also form SOCE channels in human atria as a component. TRPC1 channels can be activated by endothelin-1 or angiotensin II, which may be involved in the atrial electrical remodeling in patients with atrial fibrillation.     

Differential STIM1 expression in T and B cell subsets suggests a role in determining antigen receptor signal amplitude

Ca²⁺ acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca²⁺ entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca²⁺ concentration in the ER and an activator of the Ca²⁺ selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ∼4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca²⁺ influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca²⁺ mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses.     

Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Stromal interaction molecule 1 (STIM1) mediates Ca²⁺ movements from the extracellular space to the cytosol through a store-operated Ca²⁺ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca²⁺ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca²⁺ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca²⁺ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca²⁺ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca²⁺ movements between the SR and the cytosol.     

Store-operated Ca²+ signaling in dendritic cells occurs independently of STIM1

SOCE via CRAC channels is a critical signaling event in immune cells. Recent studies have identified key proteins underlying this process; STIM is an ER Ca2+ sensor that interacts with Orai, an intrinsic, pore-forming protein of the CRAC channel. In heterologous expression systems, STIM1 regulates SOCE by interacting with Orai1, -2, and -3. In native tissues, however, the precise roles of STIM and Orai proteins are not well defined. Here, we have investigated the molecular components of SOCE signaling in mouse DCs. We show that DCs predominantly express STIM2 and only very low levels of STIM1 compared with T lymphocytes. Upon store depletion with Tg, STIM2 aggregates and interacts selectively with Orai2. In contrast, Tg fails to aggregate STIM1 or enhance STIM1-mediated interactions with Orai proteins. Consistent with this biochemical characterization, stimulation of DCs with the adhesion molecule ICAM-1 selectively recruits STIM2 and Orai2 to the IS. Together, these data demonstrate a novel, STIM2-dependent SOCE signaling pathway in DCs.     

Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels

Agonist-activated Ca²⁺ signals in non-excitable cells are profoundly influenced by calcium entry via both store-operated and store-independent conductances. Recent studies have demonstrated that STIM1 plays a key role in the activation of store-operated conductances including the Ca²⁺-release-activated Ca²⁺ (CRAC) channels, and that Orai1 comprises the pore-forming component of these channels. We recently demonstrated that STIM1 also regulates the activity of the store-independent, arachidonic acid-regulated Ca²⁺ (ARC) channels, but does so in a manner entirely distinct from its regulation of the CRAC channels. This shared ability to be regulated by STIM1, together with their similar biophysical properties, suggested that these two distinct conductances may be molecularly related. Here, we report that whilst the levels of Orai1 alone determine the magnitude of the CRAC channel currents, both Orai1 and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orai1 increases both CRAC and ARC channel currents. Whilst similar overexpression of Orai3 alone has no effect, ARC channel currents are specifically increased by expression of Orai3 in cells stably expressing Orai1. Moreover, expression of a dominant-negative mutant Orai3, either alone or in cells expressing wild-type Orai1, profoundly and specifically reduces currents through the ARC channels without affecting those through the CRAC channels, and siRNA-mediated knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents. Importantly, our data also show that the precise effects observed critically depend on which of the three proteins necessary for effective ARC channel activity (STIM1, Orai1 and Orai3) are rate limiting under the specific conditions employed.     

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Store-operated Ca²⁺ entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca²⁺ from SR. The significant depletion of SR Ca²⁺ required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation–contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca²⁺ during periods of fibre quiescence. While Ca²⁺ in SR remains below the activation threshold for SOCE, a low-amplitude persistent Ca²⁺ influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca²⁺ and simple balancing of Ca²⁺ within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.     

Orai, STIM1 and iPLA2beta: a view from a different perspective

The mechanism of store-operated Ca²⁺ entry (SOCE) remains one of the intriguing mysteries in the field of Ca²⁺ signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca²⁺ sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA₂β as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA₂β, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA₂β in the SOCE pathway.     

Tubular aggregate myopathy and Stormorken syndrome: Mutation spectrum and genotype/phenotype correlation

Calcium (Ca²⁺) acts as a ubiquitous second messenger, and normal cell and tissue physiology strictly depends on the precise regulation of Ca²⁺ entry, storage, and release. Store‐operated Ca²⁺ entry (SOCE) is a major mechanism controlling extracellular Ca²⁺ entry, and mainly relies on the accurate interplay between the Ca²⁺ sensor STIM1 and the Ca²⁺ channel ORAI1. Mutations in STIM1 or ORAI1 result in abnormal Ca²⁺ homeostasis and are associated with severe human disorders. Recessive loss‐of‐function mutations impair SOCE and cause combined immunodeficiency, while dominant gain‐of‐function mutations induce excessive extracellular Ca²⁺ entry and cause tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). TAM and STRMK are spectra of the same multisystemic disease characterized by muscle weakness, miosis, thrombocytopenia, hyposplenism, ichthyosis, dyslexia, and short stature. To date, 42 TAM/STRMK families have been described, and here we report five additional families for which we provide clinical, histological, ultrastructural, and genetic data. In this study, we list and review all new and previously reported STIM1 and ORAI1 cases, discuss the pathomechanisms of the mutations based on the known functions and the protein structure of STIM1 and ORAI1, draw a genotype/phenotype correlation, and delineate an efficient screening strategy for the molecular diagnosis of TAM/STRMK.     

Multifaceted roles of STIM proteins

Stromal interaction molecules (STIM1 and STIM2) are critical components of store-operated calcium entry. Sensing depletion of endoplasmic reticulum (ER) Ca²⁺ stores, STIM couples with plasma membrane Orai channels, resulting in the influx of Ca²⁺ across the PM into the cytosol. Although best recognized for their primary role as ER Ca²⁺ sensors, increasing evidence suggests that STIM proteins have a broader variety of sensory capabilities than first envisaged, reacting to cell stressors such as oxidative stress, temperature, and hypoxia. Further, the array of partners for STIM proteins is now understood to range far beyond the Orai channel family. Here we discuss the implications of STIM’s expanding role, both as a stress sensor and a general modulator of multiple physiological processes in the cell.     

血管疾病治疗新靶标：STIM1和Orai1

钙离子(Ca2+)是常见的第二信使,不同于其它第二信使,其主要位于细胞外或存储于内质网(endoplasmic reticulum,ER)等细胞器内.静息状态下细胞内游离Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)约为细胞外的1/20000,受体激活或生物信号刺激可通过改变[Ca2+]i进一步发挥生物放大效应.[Ca2+]i的升高主要通过胞内Ca2+释放和胞外Ca2+内流两大途径.随着胞内钙库的排空,位于质膜上的Ca2+内流通道被激活,使Ca2+由胞外进入胞质内,这个过程称为钙库操纵的钙内流(store-operated calcium entry,SOCE),其通道称为钙库操纵的钙通道(store-operated calcium channel,SOCC).近来研究证实组成SOCC的主要蛋白是:Ca2+感受蛋白基质相互作用分子1(stromal interaction molecule 1,STIM1)[1-2]和Ca2+通道蛋白Orai1[3-4].     

The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca²⁺ signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca²⁺ sensor controlling store-operated Ca²⁺ entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca²⁺ influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca²⁺ was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy.     

ORAI1 Mutations with Distinct Channel Gating Defects in Tubular Aggregate Myopathy

Calcium (Ca²⁺) is a physiological key factor, and the precise modulation of free cytosolic Ca²⁺ levels regulates multiple cellular functions. Store‐operated Ca²⁺ entry (SOCE) is a major mechanism controlling Ca²⁺ homeostasis, and is mediated by the concerted activity of the Ca²⁺ sensor STIM1 and the Ca²⁺ channel ORAI1. Dominant gain‐of‐function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss‐of‐function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation‐dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non‐muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca²⁺ homeostasis and leading to muscle dysfunction.     

Orai1 subunit stoichiometry of the mammalian CRAC channel pore

Agonist-activated Ca2+ entry plays a critical role in Ca2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca2+ release-activated Ca2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.