ORAI Ca2+ Channels in Cancers and Therapeutic Interventions.

The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI.

Blocking Orai1 constitutive activity inhibits B-cell cancer migration and synergistically acts with drugs to reduce B-CLL cell survival.

Orai1 channel capacity to control store-operated Ca2+ entry (SOCE) and B-cell functions is poorly understood and more specifically in B-cell cancers, including human lymphoma and leukemia. As compared to normal B-cells, Orai1 is overexpressed in B-chronic lymphocytic leukemia (B-CLL) and contributes in resting B-CLL to mediate an elevated basal Ca2+ level through a constitutive Ca2+ entry, and in BCR-activated B-cell to regulate the Ca2+ signaling response. Such observations were confirmed in human B-cell lymphoma and leukemia lines, including RAMOS, JOK-1, MEC-1 and JVM-3 cells. Next, the use of pharmacological Orai1 inhibitors (GSK-7975 A and Synta66) blocks constitutive Ca2+ entry and in turn affects B-cell cancer (primary and cell lines) survival and migration, controls cell cycle, and induces apoptosis through a mitochondrial and caspase-3 independent pathway. Finally, the added value of Orai1 inhibitors in combination with B-CLL drugs (ibrutinib, idelalisib, rituximab, and venetoclax) on B-CLL survival was tested, showing an additive/synergistic effect including in the B-cell cancer lines. To conclude, this study highlights the pathophysiological role of the Ca2+ channel Orai1 in B-cell cancers, and pave the way for the use of ORAI1 modulators as a plausible therapeutic strategy.

Insights into the dynamics of the Ca2+ release-activated Ca2+ channel pore-forming complex Orai1.

An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion. The decrease in ER-luminal Ca2+ is sensed by STIM1, which undergoes structural rearrangements that lead to coupling with Orai1 and its activation. In this review, we highlight the current understanding of the Orai1 pore opening mechanism. In this context, we also point out the questions that remain unanswered and how these can be addressed by the currently emerging genetic code expansion (GCE) technology. GCE enables the incorporation of non-canonical amino acids with novel properties, such as light-sensitivity, and has the potential to provide novel insights into the structure/function relationship of CRAC channels at a single amino acid level in the living cell.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes.

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

The Role and Mechanism of STIM1/Orai1-Regulated Ca2+ Influx in Myocardial Hypertrophy in Type 2 Diabetes Mellitus.

OBJECTIVE: Diabetic cardiomyopathy (DCM) is the most common cardiovascular complication of type 2 diabetes mellitus (T2DM). Patients affected with DCM face a notably higher risk of progressing to congestive heart failure compared to other populations. Myocardial hypertrophy, a clearly confirmed pathological change in DCM, plays an important role in the development of DCM, with abnormal Ca2+ homeostasis serving as the key signal to induce myocardial hypertrophy. Therefore, investigating the mechanism of Ca2+ transport is of great significance for the prevention and treatment of myocardial hypertrophy in T2DM. METHODS: The rats included in the experiment were divided into wild type (WT) group and T2DM group. The T2DM rat model was established by feeding the rats with high-fat and high-sugar diets for three months combined with low dose of streptozotocin (100mg/kg). Afterwards, primary rat cardiomyocytes were isolated and cultured, and cardiomyocyte hypertrophy was induced through high-glucose treatment. Subsequently, mechanistic investigations were carried out through transfection with si-STIM1 and oe-STIM1. Western blot (WB) was used to detect the expression of the STIM1, Orai1 and p-CaMKII. qRT-PCR was used to detect mRNA levels of myocardial hypertrophy marker proteins. Cell surface area was detected using TRITC-Phalloidin staining, and intracellular Ca2+ concentration in cardiomyocytes was measured using Fluo-4 fluorescence staining. RESULTS: Through animal experiments, an upregulation of Orai1 and STIM1 was revealed in the rat model of myocardial hypertrophy induced by T2DM. Meanwhile, through cell experiments, it was found that in high glucose (HG)-induced hypertrophic cardiomyocytes, the expression of STIM1, Orai1, and p-CaMKII was upregulated, along with increased levels of store-operated Ca2+ entry (SOCE) and abnormal Ca2+ homeostasis. However, when STIM1 was downregulated in HG-induced cardiomyocytes, SOCE levels decreased and p-CaMKII was downregulated, resulting in an improvement in myocardial hypertrophy. To further elucidate the mechanism of action involving SOCE and CaMKII in T2DM-induced myocardial hypertrophy, high-glucose cardiomyocytes were respectively treated with BTP2 (SOCE blocker) and KN-93 (CaMKII inhibitor), and the results showed that STIM1 can mediate SOCE, thereby affecting the phosphorylation level of CaMKII and improving cardiomyocyte hypertrophy. CONCLUSION: STIM1/Orai1-mediated SOCE regulates p-CaMKII levels, thereby inducing myocardial hypertrophy in T2DM.

ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

The D84G mutation in STIM1 causes nuclear envelope dysfunction and myopathy in mice.

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor located in the sarcoplasmic reticulum (SR) of skeletal muscle, where it is best known for its role in store-operated Ca2+ entry (SOCE). Genetic syndromes resulting from STIM1 mutations are recognized as a cause of muscle weakness and atrophy. Here, we focused on a gain-of-function mutation that occurs in humans and mice (STIM1+/D84G mice), in which muscles exhibited constitutive SOCE. Unexpectedly, this constitutive SOCE did not affect global Ca2+ transients, SR Ca2+ content, or excitation-contraction coupling (ECC) and was therefore unlikely to underlie the reduced muscle mass and weakness observed in these mice. Instead, we demonstrate that the presence of D84G STIM1 in the nuclear envelope of STIM1+/D84G muscle disrupted nuclear-cytosolic coupling, causing severe derangement in nuclear architecture, DNA damage, and altered lamina A-associated gene expression. Functionally, we found that D84G STIM1 reduced the transfer of Ca2+ from the cytosol to the nucleus in myoblasts, resulting in a reduction of [Ca2+]N. Taken together, we propose a novel role for STIM1 in the nuclear envelope that links Ca2+ signaling to nuclear stability in skeletal muscle.

Dexamethasone upregulates macrophage PIEZO1 via SGK1, suppressing inflammation and increasing ROS and apoptosis.

The side effects of high-dose dexamethasone in anti-infection include increased ROS production and immune cell apoptosis. Dexamethasone effectively activates serum/glucocorticoid-regulated kinase 1 (SGK1), which upregulates various ion channels by activating store-operated calcium entry (SOCE), leading to Ca2+ oscillations. PIEZO1 plays a crucial role in macrophages' immune activity and function, but whether dexamethasone can regulate PIEZO1 by enhancing SOCE via SGK1 activation remains unclear. The effects of dexamethasone were assessed in a mouse model of sepsis, and primary BMDMs and the RAW264.7 were treated with overexpression plasmids, siRNAs, or specific activators or inhibitors to examine the relationships between SGK1, SOCE, and PIEZO1. The functional and phenotypic changes of mouse and macrophage models were detected. The results indicate that high-dose dexamethasone upregulated SGK1 by activating the macrophage glucocorticoid receptor, which enhanced SOCE and subsequently activated PIEZO1. Activation of PIEZO1 resulted in Ca2+ influx and cytoskeletal remodelling. The increase in intracellular Ca2+ mediated by PIEZO1 further increased the activation of SGK1 and ORAI1/STIM1, leading to intracellular Ca2+ peaks. In the context of inflammation, activation of PIEZO1 suppressed the activation of TLR4/NFκB p65 in macrophages. In RAW264.7 cells, PIEZO1 continuous activation inhibited the change in mitochondrial membrane potential, accelerated ROS accumulation, and induced autophagic damage and cell apoptosis in the late stage. CaMK2α was identified as a downstream mediator of TLR4 and PIEZO1, facilitating high-dose dexamethasone-induced macrophage immunosuppression and apoptosis. PIEZO1 is a new glucocorticoid target to regulate macrophage function and activity. This study provides a theoretical basis for the rational use of dexamethasone.

Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

Lead inhibits microglial cell migration via suppression of store-operated calcium entry.

Lead (Pb) is a non-biodegradable environmental pollutant that can lead to neurotoxicity by inducing neuroinflammation. Microglial activation plays a key role in neuroinflammation, and microglial migration is one of its main features. However, whether Pb affects microglial migration has not yet been elucidated. Herein, the effect of Pb on microglial migration was investigated using BV-2 microglial cells and primary microglial cells. The results showed that cell activation markers (TNF-α and CD206) in BV-2 cells were increased after Pb treatment. The migration ability of microglia was inhibited by Pb. Both store-operated calcium entry (SOCE) and the Ca2+ release-activated Ca2+ (CRAC) current were downregulated by microglia treatment with Pb in a dose-dependent manner. However, there was no statistical difference in the protein levels of stromal interaction molecule (STIM) 1, STIM2, or Ca2+ release-activated Ca2+ channel protein (Orai) 1 in microglia. The external Ca2+ influx and cell migration ability were restored to a certain extent after overexpression of either STIM1 or its CRAC activation domain in microglia. These results indicated that Pb inhibits microglial migration by downregulation of SOCE and impairment of the function of STIM1.

Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

Orai1 Ca2+ channel modulators as therapeutic tools for treating cancer: Emerging evidence!

In non-excitable cells, Orai proteins represent the main channel for Store-Operated Calcium Entry (SOCE), and also mediate various store-independent Calcium Entry (SICE) pathways. Deregulation of these pathways contribute to increased tumor cell proliferation, migration, metastasis, and angiogenesis. Among Orais, Orai1 is an attractive therapeutic target explaining the development of specific modulators. Therapeutic trials using Orai1 channel inhibitors have been evaluated for treating diverse diseases such as psoriasis and acute pancreatitis, and emerging data suggest that Orai1 channel modulators may be beneficial for cancer treatment. This review discusses herein the importance of Orai1 channel modulators as potential therapeutic tools and the added value of these modulators for treating cancer.

Development of chemical tools based on GSK-7975A to study store-operated calcium entry in cells.

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release-activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation.

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule (STIM)-gated ORAI channels at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites maintains adequate levels of Ca2+ within the ER lumen during Ca2+ signaling. Disruption of ER Ca2+ homeostasis activates the unfolded protein response (UPR) to restore proteostasis. Here, we report that the UPR transducer inositol-requiring enzyme 1 (IRE1) interacts with STIM1, promotes ER-PM contact sites, and enhances SOCE. IRE1 deficiency reduces T cell activation and human myoblast differentiation. In turn, STIM1 deficiency reduces IRE1 signaling after store depletion. Using a CaMPARI2-based Ca2+ genome-wide screen, we identify CAMKG2 and slc105a as SOCE enhancers during ER stress. Our findings unveil a direct crosstalk between SOCE and UPR via IRE1, acting as key regulator of ER Ca2+ and proteostasis in T cells and muscles. Under ER stress, this IRE1-STIM1 axis boosts SOCE to preserve immune cell functions, a pathway that could be targeted for cancer immunotherapy.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1ß, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1ß support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

An apical Phe-His pair defines the Orai1-coupling site and its occlusion within STIM1.

Ca2+ signal-generation through inter-membrane junctional coupling between endoplasmic reticulum (ER) STIM proteins and plasma membrane (PM) Orai channels, remains a vital but undefined mechanism. We identify two unusual overlapping Phe-His aromatic pairs within the STIM1 apical helix, one of which (F394-H398) mediates important control over Orai1-STIM1 coupling. In resting STIM1, this locus is deeply clamped within the folded STIM1-CC1 helices, likely near to the ER surface. The clamped environment in holo-STIM1 is critical-positive charge replacing Phe-394 constitutively unclamps STIM1, mimicking store-depletion, negative charge irreversibly locks the clamped-state. In store-activated, unclamped STIM1, Phe-394 mediates binding to the Orai1 channel, but His-398 is indispensable for transducing STIM1-binding into Orai1 channel-gating, and is spatially aligned with Phe-394 in the exposed Sα2 helical apex. Thus, the Phe-His locus traverses between ER and PM surfaces and is decisive in the two critical STIM1 functions-unclamping to activate STIM1, and conformational-coupling to gate the Orai1 channel.