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Background/Aims: Upon Ca2+ store depletion, stromal interaction molecule 1 (STIM1) oligomerizes, redistributes near plasmalemma to interact with Ca2+ selective channel-forming subunit (Orai1) and initiates store-operated Ca2+ entry (SOCE). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a regulator of SOCE, but how CaMKII regulates SOCE remains obscure. Methods: Using Fura2, confocal microscopy, co-immunoprecipitation, specific blocker and overexpression/knockdown approaches, we evaluated STIM1 aggregation and its interaction with Orai1, and SOCE upon Ca2+ store depletion in thapsigargin (TG) treated HEK293 and HeLa cells. Results: Overexpression of CaMKIIδ enhanced TG-induced STIM1 co-localization and interaction with Orai1 as well as SOCE. In contrast, CaMKIIδ knockdown and a specific inhibitor of CaMKII suppressed them. In addition, overexpression or knockdown of CaMKIIδ in TG treated cells exhibited increased or reduced STIM1 clustering and plasmalemma redistribution, respectively. Conclusion: CaMKII up-regulates SOCE by increasing STIM1 aggregation and interaction with Orai1. This study provides an additional insight into SOCE regulation and a potential mechanism for CaMKII involvement in some pathological situations through crosstalk with SOCE.

The dysregulation of store-operated Ca2+ entry (SOCE) promotes cancer progression by changing Ca2+ levels in the cytosol or endoplasmic reticulum. Stromal interaction molecule 1 (STIM1), a component of SOCE, is upregulated in several types of cancer and responsible for cancer cell migration, invasion, and metastasis. To explore the impact of STIM1-mediated SOCE on the turnover of focal adhesion (FA) and cell migration, we overexpressed the wild-type and constitutively active or dominant negative variants of STIM1 in an osteosarcoma cell line. In this study, we hypothesized that STIM1-mediated Ca2+ elevation may increase cell migration. We found that constitutively active STIM1 dramatically increased the Ca2+ influx, calpain activity, and turnover of FA proteins, such as the focal adhesion kinase (FAK), paxillin, and vinculin, which impede the cell migration ability. In contrast, dominant negative STIM1 decreased the turnover of FA proteins as its wild-type variant compared to the cells without STIM1 overexpression while promoting cell migration. These unexpected results suggest that cancer cells need an appropriate amount of Ca2+ to control the assembly and disassembly of focal adhesions by regulating calpain activity. On the other hand, overloaded Ca2+ results in excessive calpain activity, which is not beneficial for cancer metastasis.

Store-operated Ca2+ entry (SOCE) is a ubiquitous and essential mechanism regulating Ca2+ homeostasis in all tissues, and controls a wide range of cellular functions including keratinocyte differentiation, osteoblastogenesis and osteoclastogenesis, T cell proliferation, platelet activation, and muscle contraction. The main SOCE actors are STIM1 and ORAI1. Depletion of the reticular Ca2+ stores induces oligomerization of the luminal Ca2+ sensor STIM1, and the oligomers activate the plasma membrane Ca2+ channel ORAI1 to trigger extracellular Ca2+ entry. Mutations in STIM1 and ORAI1 result in abnormal SOCE and lead to multi-systemic disorders. Recessive loss-of-function mutations are associated with CRAC (Ca2+ release-activated Ca2+) channelopathy, involving immunodeficiency and autoimmunity, muscular hypotonia, ectodermal dysplasia, and mydriasis. In contrast, dominant STIM1 and ORAI1 gain-of-function mutations give rise to tubular aggregate myopathy and Stormorken syndrome (TAM/STRMK), forming a clinical spectrum encompassing muscle weakness, thrombocytopenia, ichthyosis, hyposplenism, short stature, and miosis. Functional studies on patient-derived cells revealed that CRAC channelopathy mutations impair SOCE and extracellular Ca2+ influx, while TAM/STRMK mutations induce excessive Ca2+ entry through SOCE over-activation. In accordance with the opposite pathomechanisms underlying both disorders, CRAC channelopathy and TAM/STRMK patients show mirror phenotypes at the clinical and molecular levels, and the respective animal models recapitulate the skin, bones, immune system, platelet, and muscle anomalies. Here we review and compare the clinical presentations of CRAC channelopathy and TAM/STRMK patients and the histological and molecular findings obtained on human samples and murine models to highlight the mirror phenotypes in different tissues, and to point out potentially undiagnosed anomalies in patients, which may be relevant for disease management and prospective therapeutic approaches. [ABSTRACT FROM AUTHOR]

Introduction: Rac1 and STIM1 genes are emerging therapeutic targets for cancers. However, their roles in acute myeloid leukaemia (AML) are not well understood. The goal of this study was to evaluate the effects of dose and time on Rac1 and STIM1 knockdown in the AML cell line model (THP-1 cells). Methods: THP-1 cells were transfected with siRac1 at doses of 50, 100, and 200 nM or dsiSTIM1 at doses of 2, 5, and 10 nM. Expression level of Rac1 and STIM1 then were assessed at time points between 12 and 72 h post-transfection using real-time reverse transcription polymerase chain reaction. Results: Compared to the control, 87% Rac1 knockdown was attained with 50 nM siRac1 at 24 h post-transfection, and 70% STIM1 knockdown was achieved with 10 nM dsiSTIM1 at 48 h post-transfection. Conclusion: These results show that effective knockdown of Rac1 and STIM1 is possible, and therapy that includes Rac1 and STIM1 inhibitors eventually could provide a new and highly effective strategy for AML treatment. [ABSTRACT FROM AUTHOR]

Predictive value of serum complement Clq tumor necrosis factor-related protein 9 (CTRP9) and serum stromal interaction molecule 1 (STIM1) was investigated for restenosis after cerebrovascular stent implantation, as well as its relationship with vasoactive substances and inflammatory cytokines. In this prospective study, 128 patients with cerebral infarction treated with cerebrovascular stent implantation in Yantaishan Hospital were recruited. A total of 66 cases with restenosis after cerebrovascular stent implantation were included in group A, and 62 cases without stenosis were included in group B. Serum CTRP9 and STIM1 levels were measured by enzyme-linked immunosorbent assay (ELISA). ROC curves of serum CTRP9 and STIM1 levels in patients with postoperative restenosis were drawn. The vasoactive substances nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were analyzed by ELISA. The correlation of serum CTRP9, STIM1 levels and NO, TNF-α, IL-6 were analyzed by Pearson correlation coefficient. Serum CTRP9 and NO levels in group A were significantly lower than those in group B. The levels of serum STIM1, TNF-α and IL-6 in group A were significantly higher than those in group B (P<0.001). The sensitivity and specificity of serum CTRP9 level in the diagnosis of restenosis after cerebrovascular stent implantation were, respectively, 59.68 and 75.76%. Those of serum STIM1 were, respectively, 87.10 and 46.97% and those of the combination of serum CTRP9 and STIM1 were 90.32 and 48.48%. Serum CTRP9 level was positively correlated with NO, and negatively correlated with TNF-α and IL-6. STIM1 was positively correlated with TNF-α and IL-6, and negatively correlated with NO (P<0.001). Serum CTRP9 level was significantly decreased in patients with restenosis after cerebrovascular stent implantation, while STIM1 level was significantly up-regulated. Both were correlated with the change of NO, IL-6 and TNF-α levels, therefore they could be used as biological indicators for prediction of restenosis after cerebrovascular stent implantation. [ABSTRACT FROM AUTHOR]

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell‐specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non‐pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell‐mediated inflammatory diseases. Synopsis: Pathogenic Th17 cells have been implicated in autoimmune diseases, while non‐pathogenic Th17 cells provide immunity to fungal pathogens. Patients with mutations in ORAI1 or STIM1 have impaired Ca2+ signaling in immune cells and are more susceptible to infections with fungal pathogens. A novel missense mutation in STIM1 (p.L374P) abolishes Ca2+ signals in immune cells by interfering with the activation of ORAI1, the pore‐forming subunit of the calcium release‐activated calcium (CRAC) channel.T cells of patients with STIM1 p.L374P mutation fail to produce cytokines when challenged with C. albicans and have severe defects in metabolic functions including glycolysis and oxidative phosphorylation (OXPHOS).Deletion of STIM1 and its homologue STIM2 in all immune cells results in enhanced severity of mucosal C. albicans infection, which is associated with defective T cell and neutrophil function.T cell‐specific deletion of STIM1 reduces resistance to systemic C. albicans infection and is associated with impaired effector functions of Th1 and non‐pathogenic Th17 cells.STIM1 is required for the transcriptional regulation of aerobic glycolysis and OXPHOS in non‐pathogenic Th17 cells, whereas glycolysis in pathogenic Th17 cells is independent of STIM1 and CRAC channel function. [ABSTRACT FROM AUTHOR]

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 μM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 μM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs. [ABSTRACT FROM AUTHOR]

Background: Stromal interaction molecule 1 (STIM1) is one of the main components of the store operated Ca2+ entry (SOCE) signaling pathway. Individuals with mutated STIM1 present severely hypomineralized enamel characterized as amelogenesis imperfecta (AI) but the downstream molecular mechanisms involved remain unclear. Circadian clock signaling plays a key role in regulating the enamel thickness and mineralization, but the effects of STIM1 -mediated AI on circadian clock are unknown. Objectives: The aim of this study is to examine the potential links between SOCE and the circadian clock during amelogenesis. Methods: We have generated mice with ameloblast-specific deletion of Stim1 (Stim1 fl/fl/Amelx-iCre+/+, Stim1 cKO) and analyzed circadian gene expression profile in Stim1 cKO compared to control (Stim1 fl/fl/Amelx-iCre–/–) using ameloblast micro-dissection and RNA micro-array of 84 circadian genes. Expression level changes were validated by qRT-PCR and immunohistochemistry. Results: Stim1 deletion has resulted in significant upregulation of the core circadian activator gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocation 1 (Bmal1) and downregulation of the circadian inhibitor Period 2 (Per2). Our analyses also revealed that SOCE disruption results in dysregulation of two additional circadian regulators; p38α mitogen-activated protein kinase (MAPK14) and transforming growth factor-beta1 (TGF-β1). Both MAPK14 and TGF-β1 pathways are known to play major roles in enamel secretion and their dysregulation has been previously implicated in the development of AI phenotype. Conclusion: These data indicate that disruption of SOCE significantly affects the ameloblasts molecular circadian clock, suggesting that alteration of the circadian clock may be partly involved in the development of STIM1 -mediated AI. [ABSTRACT FROM AUTHOR]

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromalinteracting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate storeoperated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletionindependent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated. [ABSTRACT FROM AUTHOR]

Summary: Obinutuzumab is thought to exert its effects through its high antibody‐dependent cellular cytotoxicity (ADCC) via glyco‐engineering of the Fc region. In addition, obinutuzumab causes direct binding‐induced cell death (DCD) only by specifically binding to its target CD20, a Ca2+ channel. However, the specific features of CD20 related to obinutuzumab binding‐induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca2+ channel features of CD20 as a store‐operated Ca2+ channel (SOC) and obinutuzumab binding‐induced cell death. Ca2+ channel function and biochemical analysis revealed that CD20 is an Orai1‐ and stromal interaction molecule (STIM1)‐dependent Ca2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton‐X solubilized obinutuzumab‐bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock‐down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton‐soluble membrane region. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is a unique head and neck malignancy with highly metastatic cell-biological characteristics, for which latent EBV-infection is responsible. Our earlier studies showed that EGF-stimulated Ca2+ signaling via store-operated Ca2+ entry (SOCE) was amplified in NPC cells expressing EBV-encoded LMP1, thus contributing to EBV-enhanced metastatic capacities. However, the pathway through which EBV modulates cytosolic Ca2+ signaling still remains unclear. Here, we demonstrated that EBV-infection amplified EGF-stimulated Ca2+ responses through the promotion of intracellular aggregation of STIM1, which serves as a Ca2+ sensor to activate SOCE. Blockage of EBV-remodeled Ca2+ signaling by STIM1-silencing inhibited cell migration by interrupting epithelial-mesenchymal transition (EMT) in vitro, and suppressed tumor dissemination in zebrafish and lymph node metastasis in mice. In addition, STIM1 expression was upregulated in primary NPC tissues compared with normal nasopharyngeal epithelium and stronger among the patients with advanced lymph node metastatic disease (N2-3 stage). Our findings thus indicate that EBV promotes metastatic potential by enhancing STIM1-dependent Ca2+ signaling that manipulates EMT in NPC cells. EBV-modulated Ca2+ signaling could serve as a candidate anti-metastatic target for NPC treatment. [ABSTRACT FROM AUTHOR]

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T‐cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T‐cell activation. Hence, TCR engagement of EGR4−/− T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti‐tumor immunity in EGR4−/− mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T‐cell differentiation and function. Synopsis: EGR proteins regulate T cell development and differentiation, but no role for EGR4 has previously been shown. T cell activation upregulates EGR4, whereas EGR4−/− T cells exhibit increased Ca2+ responses, promoting Th1 bias and anti‐cancer immunity. EGR4 is expressed in activated T cells.EGFR4 suppresses Ca2+ responses by regulating KCa3.1 and Kv1.3 expression.In the absence of EGR4, Ca2+‐dependent NFAT activation drives Th1 differentiation.EGR4−/− T cells exhibit enhanced anti‐cancer immunity. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is a disease with poor prognosis which urgently is in need of effective prognostic marker. To discover novel prognostic protein marker for ESCC, we applied a high-throughput monoclonal antibody microarray to compare tumor and adjacent non-tumor tissues from ESCC patients. Antibody #ESmAb270 was consistent higher expressed in tumors and it was identified via mass spectrometry to be stromal interaction molecule 1 (STIM1). STIM1 H scores in tumor tissues were significantly up-regulated in esophageal tumor tissues compared to non-tumor tissues in 105 ESCC patients. We also observed that high STIM1 expression was correlated with advanced tumor grade and poor prognosis of ESCC. In addition, attenuation of STIM1 by siRNA or chemical inhibitors significantly inhibited cell viability and migration of ESCC cells. Evidence from high-throughput monoclonal antibody microarray, IHC microarray with associated survival data and functional analysis show that STIM1 is an unfavorable prognostic biomarker in ESCC. • Antibody microarray is a unique approach to identify differentially expressed proteins. • STIM1 was found to be up-regulated in esophageal squamous cell carcinoma. • STMI1 expression is associated with poor survival in esophageal squamous cell carcinoma. • Attenuation of STIM1 by siRNA inhibited cell viability and migration of esophageal squamous cell carcinoma cells. [ABSTRACT FROM AUTHOR]

Store‐operated Ca2+ entry through Orai1 channels is a primary mechanism for Ca2+ entry in many cells and mediates numerous cellular effector functions ranging from gene transcription to exocytosis. Orai1 channels are amongst the most Ca2+‐selective channels known and are activated by direct physical interactions with the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) in response to store depletion triggered by stimulation of a variety of cell surface G‐protein coupled and tyrosine kinase receptors. Work in the last decade has revealed that the Orai1 gating process is highly cooperative and strongly allosteric, likely driven by a wave of interdependent conformational changes throughout the protein originating in the peripheral C‐terminal ligand binding site and culminating in pore opening. In this review, we survey the structural and molecular features in Orai1 that contribute to channel gating and consider how they give rise to the unique biophysical fingerprint of Orai1 currents. [ABSTRACT FROM AUTHOR]

In chronic kidney disease, renal phosphate retention leads to hyperphosphatemia with subsequent vascular osteogenic signaling and calcification. Osteogenic signaling involves up-regulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme stimulating calcification by degrading the calcification inhibitor pyrophosphate. Stimulation of osteogenic signaling and calcification by phosphate donor β-glycerophosphate in human aortic smooth muscle cells (HAoSMCs) is attenuated by MgCl 2 , an effect mimicked by Ca2+-sensing receptor agonist GdCl 3. Most recent observations revealed that the effect of β-glycerophosphate on osteogenic signaling requires ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE), which is stimulated by Ca2+-sensor STIM1. The present study explored whether ORAI1 and/or STIM1 expression and, thus, SOCE and osteogenic signaling in HAoSMCs are sensitive to MgCl 2 and/or GdCl 3. To this end, transcript levels were estimated using q-RT-PCR, protein abundance with western blotting, cytosolic Ca2+-concentration ([Ca2+] i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+] i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 μM). As a result, 24 h exposure to β-glycerophosphate (2 mM) significantly enhanced transcript levels of ORAI1 and STIM1 as well as SOCE, effects significantly blunted or virtually abrogated by 1.5 mM MgCl 2 and by 50 μM GdCl 3. In conclusion, MgCl 2 and GdCl 3 are powerful inhibitors of ORAI1 and STIM1 expression and store-operated Ca2+-entry, effects affecting osteogenic signalling in vascular smooth muscle cells. • MgCl 2 and CaSR-agonist GdCl 3 abrogate phosphate-induced Orai1+STIM1 expression. • MgCl 2 and GdCl 3 abrogate phosphate-induced store operated Ca2+-entry (SOCE). • MgCl 2 -and GdCl 3 -sensitive Orai1/STIM1/SOCE contribute to vascular calcification. [ABSTRACT FROM AUTHOR]

Background/Aims: Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+- sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. Methods: The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. Results: Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. Conclusion: Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC. [ABSTRACT FROM AUTHOR]

Gemcitabine (GEM) alone and GEM‐based chemotherapy are the preferred regimens for treating advanced unresectable and metastatic pancreatic cancer (PC). However, these treatments have limited efficacy due to acquired resistance of cancer cells to chemotherapy, the mechanisms of which are not fully understood. In this study, we established two stable multidrug‐resistant cell lines, BxPC‐3‐GR and CFPAC‐1‐GR, from their corresponding parental cells through exposure to GEM following a stepwise incremental dosing strategy. The GEM IC50 values of BxPC‐3‐GR and CFPAC‐1‐GR increased 112‐fold and 210‐fold, respectively, compared to parental cell lines. In vitro and in vivo experiments confirmed that both GEM‐resistant cell subgroups declined in proliferative capacity, but were more resistant to GEM. Unlike CFPAC‐1‐GR, BxPC‐3‐GR exhibited enhanced migratory and invasive properties compared with BxPC‐3 in vitro. We also compared differentially expressed mRNA profiles between parental and GEM‐resistant cells using transcriptome sequencing. RRM1, STIM1, and TRIM21 were significantly upregulated in both GEM‐resistant cell lines and confirmed to be associated with the degree of GEM resistance by quantitative reverse‐transcription polymerase chain reaction and western blot analysis. These three genes were more highly expressed in PC tissues and potentially regarded as prognostic biomarkers through database mining. Thus, our findings provide chemo‐resistant cell models to better understand the underlying mechanisms of chemoresistance, and to explore potential biomarkers for GEM response in PC patients. [ABSTRACT FROM AUTHOR]

In vascular smooth muscle cells (VMSCs), the stimulation of store-operated channels (SOCs) mediate Ca2+ influx pathways which regulate important cellular functions including contraction, proliferation, migration, and growth that are associated with the development of vascular diseases. It is therefore important that we understand the biophysical, molecular composition, activation pathways, and physiological significance of SOCs in VSMCs as these maybe future therapeutic targets for conditions such as hypertension and atherosclerosis. Archetypal SOCs called calcium release-activated channels (CRACs) are composed of Orai1 proteins and are stimulated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) following store depletion. In contrast, this review focuses on proposals that canonical transient receptor potential (TRPC) channels composed of a heteromeric TRPC1/C5 molecular template, with TRPC1 conferring activation by store depletion, mediate SOCs in native contractile VSMCs. In particular, it summarizes our recent findings which describe a novel activation pathway of these TRPC1-based SOCs, in which protein kinase C (PKC)-dependent TRPC1 phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) are obligatory for channel opening. This PKC- and PIP2-mediated gating mechanism is regulated by the PIP2-binding protein myristoylated alanine-rich C kinase (MARCKS) and is coupled to store depletion by TRPC1-STIM1 interactions which induce Gq/PLCβ1 activity. Interestingly, the biophysical properties and activation mechanisms of TRPC1-based SOCs in native contractile VSMCs are unlikely to involve Orai1. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario. [ABSTRACT FROM AUTHOR]

Whereas cardiac TRPC (transient receptor potential canonical) channels and the associated store-operated Ca2+ entry (SOCE) are abnormally elevated during cardiac hypertrophy and heart failure, the mechanism of this upregulation is not fully elucidated but might be related to the activation of the mineralocorticoid pathway. Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 μM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitivemacroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to determine whether STIM1 is involved in the regulation of cellular movement in the promotion of osteosarcoma metastasis. Methods: The expression of STIM1 was silenced by si RNA targeting STIM1 in MG-63 osteosarcoma cells. Then the invasion experiment, migration experiment and adhesion experiment were used to detect the changes in the ability of invasion, migration and adhesion of osteosarcoma cells. The expression of FAK and paxillin, as well as the activity of RhoA and Rac1 signaling pathway were detected by Western Blot. Results: The expression and transcription of STIM1 in MG-63 osteosarcoma cells were significantly reduced after we transfected si RNA targeting STIM1(P<0.05). Then, we found that the invasion, migration and adhesion abilities of MG-63 osteosarcoma cells were significantly decreased after silencing STIM1 expression in MG-63 osteosarcoma cells(P<0.05). In addition, the levels of FAK and paxillin, important components of cellular pseudopod and cytoskeleton, were also significantly lowered(P<0.05). Further studies showed that silencing STIM1 expression in MG-63 osteosarcoma cells significantly inhibited the activity of RhoA and Rac1 signaling pathways(P<0.05). Conclusion: STIM1 activates RhoA and Rac1 signaling pathways to increase expression of FAK and paxillin, thus regulate osteosarcoma cell movement and promote osteosarcoma metastasis. [ABSTRACT FROM AUTHOR]

Background: Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. Methods: An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal CaC levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. Results: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. Conclusions: These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane. [ABSTRACT FROM AUTHOR]

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta-structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1-ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca(2+) influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1-ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P(2) depletion.

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. [Display omitted] • IRE1 promotes SOCE, T cell activation, and muscular differentiation via STIM1 • STIM1 supports IRE1 activity upon store-depletion-mediated ER stress • CaMPARI screening identifies CAMK2G and SLC1A5 as SOCE boosters under early ER stress Carreras-Sureda et al. report that IRE1 protein facilitates STIM1 to gate ORAI channels in ER-PM contact sites and SOCE, supporting T cell activation and myoblast differentiation. Noteworthily, STIM1 promotes IRE1 signaling upon store depletion. Finally, whole-genome CaMPARI screening identifies CAMK2G and SLC1A5 as ER-stress-induced SOCE enhancers. [ABSTRACT FROM AUTHOR]

Pneumonia is a disease caused by inflammation and has high morbidity and mortality rates. Stromal interaction molecule 1 (STIM1) is involved in the regulation of inflammatory processes. However, to the best of the authors' knowledge, the role of STIM1 in pneumonia has not yet been reported. In the present study, lipopolysaccharide (LPS) was administered to A549 cells to construct a cell damage model. The expression of STIM1 in the model cells was detected by western blotting and reverse transcription-quantitative PCR. Then, STIM1 expression was inhibited and cell survival was detected by Cell Counting Kit-8 and flow cytometry. The expression of inflammatory factors was detected by enzyme-linked immunosorbent assay and endoplasmic reticulum stress (ERS)-related proteins were detected by immunofluorescence and western blotting. Subsequently, the relationship between insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) and STIM1 was verified by RNA-binding protein immunoprecipitation assay and actinomycin D treatment. Finally, the regulatory mechanism of IGF2BP2 and STIM1 in LPS-induced A549 cells was further investigated. The results of the present study demonstrated that STIM1 expression was increased in LPS-induced A549 cells and that STIM1 knockdown inhibited LPS-induced A549 cell apoptosis and alleviated LPS-induced A549 cell inflammation and ERS. In addition, IGF2BP2 enhanced the stability of STIM1 mRNA and knockdown of IGF2BP2-regulated STIM1 expression alleviated LPS-induced ERS and inflammatory responses in A549 cells. In conclusion, knockdown of IGF2BP2-regulated STIM1 improved cell damage in the LPS-induced pneumonia cell model by alleviating ERS and the inflammatory response. [ABSTRACT FROM AUTHOR]

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca2+ levels and activates Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 that lead to cytoskeletal remodeling are not known. The proximity-labeling technique (BioID) can capture weak and transient protein-protein interactions, including proteins that reside in the close vicinity of the bait, but that may not be direct binders. Hence, in the present study, we investigated the STIM1 interactome using the BioID technique. A promiscuous biotin ligase was fused to the cytoplasmic C-terminus of STIM1 and was stably expressed in a mouse embryonic fibroblast (MEF) cell line. Screening of biotinylated proteins identified several high confidence targets. Here, we report Gelsolin (GSN) as a new member of the STIM1 interactome. GSN is a Ca2+-dependent actin-severing protein that promotes actin filament assembly and disassembly. Results were validated using knockdown approaches and immunostaining. We tested our results in neonatal cardiomyocytes where STIM1 overexpression induced altered actin dynamics and cytoskeletal instability. This is the first time that BioID assay was used to investigate the STIM1 interactome. Our work highlights the role of STIM1/GSN in the structure and function of the cytoskeleton.

Swift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP3R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level.

Intracellular Ca2+ ions represent a signaling mediator that plays a critical role in regulating different muscular cellular processes. Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.

Calcium (Ca2+) signaling plays a dichotomous role in cellular biology, controlling cell survival and proliferation on the one hand and cellular toxicity and cell death on the other. Store-operated Ca2+ entry (SOCE) by CRAC channels represents a major pathway for Ca2+ entry in non-excitable cells. The CRAC channel has two key components, the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM) and the plasma-membrane Ca2+ channel Orai. Physical coupling between STIM and Orai opens the CRAC channel and the resulting Ca2+ flux is regulated by a negative feedback mechanism of slow Ca2+ dependent inactivation (SCDI). The identification of the SOCE-associated regulatory factor (SARAF) and investigations of its role in SCDI have led to new functional and molecular insights into how SOCE is controlled. In this review, we provide an overview of the functional and molecular mechanisms underlying SCDI and discuss how the interaction between SARAF, STIM1, and Orai1 shapes Ca2+ signaling in cells.

Interaction between the endoplasmic reticulum protein STIM1 and the plasma membrane channel ORAI1 generates calcium signals that are central for diverse cellular functions. How STIM1 binds and activates ORAI1 remains poorly understood. Using electrophysiological, optical, and biochemical techniques, we examined the effects of mutations in the STIM1-ORAI1 activating region (SOAR) of STIM1. We find that SOAR mutants that are deficient in binding to resting ORAI1 channels are able to bind to and boost activation of partially activated ORAI1 channels. We further show that the STIM1 binding regions on ORAI1 undergo structural rearrangement during channel activation. The results suggest that activation of ORAI1 by SOAR occurs in multiple steps. In the first step, SOAR binds to ORAI1, partially activates the channel, and induces a rearrangement in the SOAR-binding site of ORAI1. That rearrangement of ORAI1 then permits sequential steps of SOAR binding, via distinct molecular interactions, to fully activate the channel.

Shank3, a key molecule related to the development and deterioration of autism, has recently been found to downregulate in the murine brain after ischemia/reperfusion (I/R). Despite this discovery, however, its effects on neuronal injury and the mechanism underlying the effects remain to be clarified. To address this, in this study, based on genetically modified mice models, we revealed that the expression of Shank3 showed a time-dependent change in murine hippocampal neurons after I/R, and that conditional knockout (cko) of Shank3 in neurons resulted in aggravated neuronal injuries. The protective effects of Shank3 against oxidative stress and inflammation after I/R were achieved through direct binding STIM1 and subsequent proteasome-mediated degradation of STIM1. The STIM1 downregulation induced the phosphorylation of downstream Nrf2 Ser40, which subsequently translocated to the nucleus, and further increased the expression of antioxidant genes such as NQO1 and HO-1 in HT22 cells. In vivo, the study has further confirmed that double knockout of Shank3 and Stim1 alleviated oxidative stress and inflammation after I/R in Shank3 cko mice. In conclusion, the present study has demonstrated that Shank3 interacts with STIM1 and inhibits post-I/R neuronal oxidative stress and inflammatory response via the Nrf2 pathway. This interaction can potentially contribute to the development of a promising method for I/R treatment., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)