Loss of Stim2 in zebrafish induces glaucoma-like phenotype.

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5 days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology.

STIM1 functions as a proton sensor to coordinate cytosolic pH with store-operated calcium entry.

The meticulous regulation of intracellular pH (pHi) is crucial for maintaining cellular function and homeostasis, impacting physiological processes such as heart rhythm, cell migration, proliferation, and differentiation. Dysregulation of pHi is implicated in various pathologies such as arrhythmias, cancer, and neurodegenerative diseases. Here, we explore the role of STIM1, an ER calcium (Ca2+) sensor mediating Store Operated Ca2+ Entry (SOCE), in sensing pHi changes. Our study reveals that STIM1 functions as a sensor for pHi changes, independent of its Ca2+-binding state. Through comprehensive experimental approaches including confocal microscopy, FRET-based sensors, and mutagenesis, we demonstrate that changes in pHi induce conformational alterations in STIM1, thereby modifying its subcellular localization and activity. We identify two conserved histidine within STIM1 essential for sensing pHi shifts. Moreover, intracellular alkalization induced by agents such as Angiotensin II or NH4Cl enhances STIM1-mediated SOCE, promoting cardiac hypertrophy. These findings reveal a novel facet of STIM1 as a multi-modal stress sensor that coordinates cellular responses to both Ca2+ and pH fluctuations. This dual functionality underscores its potential as a therapeutic target for diseases associated with pH and Ca2+ dysregulation.

A Deep Dive into the N-Terminus of STIM Proteins: Structure-Function Analysis and Evolutionary Significance of the Functional Domains.

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca2+ levels ([Ca2+]i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca2+]i through store-operated Ca2+ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca2+]ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca2+ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins.

High cadmium exposure impairs adult hippocampal neurogenesis via disruption of store-operated calcium entry.

Cadmium (Cd) is a neurotoxicant that gradually accumulates in the human body with age. High Cd burden is correlated with adult hippocampal neurogenesis (AHN) and memory deficits in mammals. However, little knowledge is known about the mechanism by which Cd exposure impairs neurogenesis and cognition. Here, we investigated the roles of store-operated calcium entry (SOCE)-mediated calcium dyshomeostasis in Cd-induced AHN and memory deficits as well as therapeutic potential for the prevention of Cd-induced neurotoxicity. To achieve this goal, 8 weeks-old C57BL/6 J mice were subjected to different concentrations of cadmium chloride (0, 5, 10, 20 ppm) in drinking water for 8 weeks, we then examined the AHN, calcium homeostasis, SOCE channel and memory in Cd-exposed mice by using immunohistochemistry, calcium imaging, Y-maze and fear conditioning test. Our results indicated that chronic Cd exposure markedly increased Cd levels in serum and cerebrospinal fluid by almost 10-fold, and inhibited the proliferation and differentiation of hippocampal adult neural stem cells in a dose-dependent manner. Additionally, Cd exposure impaired the maturation of hippocampal neural stem cells without inducing gliosis. Transcriptome analysis revealed that Cd exposure inhibited the proliferation of neuroblastoma via alteration of calcium signaling pathway, and attenuated SOCE channels played a pivotal role in mediating Cd-induced cytoplasmic calcium overload and depletion of endoplasmic reticulum calcium stores. Activation of SOCE by hyperforin, a natural derivative from medicinal plant, restored intracellular calcium homeostasis and improved AHN and memory in Cd-exposed mice. Together, this study provided novel insights into the mechanism that Cd exposure impaired AHN and memory by prompting neuronal SOCE-mediated calcium dyshomeostasis, and offered a new therapeutic approach for prevention of Cd-induced neurotoxicity.

Fatty acids promote migration of CD4+ T cells through calcium release-activated calcium modulator ORAI1 sensitive glycolysis in dairy cows.

Nutritional and metabolic state in dairy cows are important determinants of the immune response. During the periparturient period, a state of negative energy balance in the cow increases plasma concentrations of fatty acids, which are associated with inflammation. Among immune cells, CD4+ T are able to function under high fatty acid (High-FA) conditions, but the underlying mechanisms regulating these events remain unclear. The objective of this study was to clarify the functional mechanisms of CD4+ T cells under High-FA conditions. Effects of glycolysis and calcium release-activated calcium modulator 1 (ORAI1) on migration of CD4+ T cells exposed to High-FA were investigated in vivo and in vitro. CD4+ T cells were isolated from peripheral blood of healthy (n = 9) and High-FA (n = 9) Holstein cows (average 2.5 ± 0.2 lactations, 12.3 ± 0.8 d in milk). In the first experiment, real-time quantitative PCR (RT-qPCR) was used to assess chemokine receptors in isolated CD4+ T cells and migration capacity. The relative mRNA measurements results revealed downregulation of CCR1 and CXCR2, and upregulation of CCR2, CCR4, CCR5, CCR7, CCR8, CCR10, CXCR1, CXCR3, CXCR4, CX3CR1. Among them, the expression of CXCR4 was relatively high. Therefore, CXCL12, a ligand chemokine of CXCR4, was an inducer of CD4+ T cell migration. CD4+ T cells were inoculated in the upper chamber and CXCL12 (100 ng/mL, Peprotech) in RPMI1640 was added to the lower chamber and transmigrate for 3 h at 37°C and 5% CO2. The cell migration assay revealed that migration capacity of CD4+ T cells from High-FA cows was greater. RT-qPCR indicated greater abundance of the glycolysis-related targets HIF1A, HK2, PKM2, Glut1, GAPDH, LDHA and Western blotting indicated greater abundance of the glycolysis-related targets HIF1A, HK2, PKM2, Glut1, GAPDH and LDHA in CD4+ T cells of High-FA cows. To characterize specific mechanisms of CD4+ T cell migration in vitro, cells from the spleens of 3 newborn healthy female Holstein calves were isolated (1 d old, 40-50 kg) after euthanasia. Inhibition of glycolysis attenuated the migration ability of cells, but had no effect on the protein and mRNA abundance of SOCE-associated ORAI1 and STIM1. In contrast, ORAI1 was upregulated in CD4+ T cells of cows exposed to high-FA. To explore the potential mechanisms whereby an active glycolytic metabolism affects CD4+ T cells under High-FA conditions, we knocked down ORAI1 (siORAI1) using small interfering RNA. Isolated CD4+ T cells from High-FA cows with the siORAI1 had an attenuated glycolytic metabolism and migration capacity. Taken together, these data suggested that calcium ions in CD4+ T cells from cows with High-FA regulate glycolytic metabolism and influence cell migration at least in part by modulating ORAI1. Thus, these studies identified a novel mechanism of Ca2+ regulation of CD4+ T cell glycolytic metabolism affecting their migration through the SOCE pathway.

Impaired calcium influx underlies skewed T helper cell differentiation in children with IgE-mediated food allergies.

BACKGROUND: Reasons for Th2 skewing in IgE-mediated food allergies remains unclear. Clinical observations suggest impaired T cell activation may drive Th2 responses evidenced by increased atopic manifestations in liver transplant patients on tacrolimus (a calcineurin inhibitor). We aimed to assess differentiation potential, T cell activation and calcium influx of naïve CD4+ T cells in children with IgE-mediated food allergies. METHODS: Peripheral blood mononuclear cells from infants in the Starting Time for Egg Protein (STEP) Trial were analyzed by flow cytometry to assess Th1/Th2/Treg development. Naïve CD4+ T cells from children with and without food allergies were stimulated for 7 days to assess Th1/Th2/Treg transcriptional factors and cytokines. Store operated calcium entry (SOCE) was measured in children with and without food allergies. The effect of tacrolimus on CD4+ T cell differentiation was assessed by treating stimulated naïve CD4+ T cells from healthy volunteers with tacrolimus for 7 days. RESULTS: Egg allergic infants had impaired development of IFNγ+ Th1 cells and FoxP3+ transitional CD4+ T cells compared with non-allergic infants. This parallels reduced T-bet, IFNγ and FoxP3 expression in naïve CD4+ T cells from food allergic children after in vitro culture. SOCE of naïve CD4+ T cells was impaired in food allergic children. Naïve CD4+ T cells treated with tacrolimus had reduced IFNγ, T-bet, and FoxP3, but preserved IL-4 expression. CONCLUSIONS: In children with IgE-mediated food allergies, dysregulation of T helper cell development is associated with impaired SOCE, which underlies an intrinsic impairment in Th1 and Treg differentiation. Along with tacrolimus-induced Th2 skewing, this highlights an important role of SOCE/calcineurin pathway in T helper cell differentiation.

Blockade of store-operated calcium entry by BTP2 preserves anti-inflammatory gene expression in human peripheral blood mononuclear cells.

Store-operated calcium entry (SOCE) is essential for cellular signaling. Earlier studies of the pyrazole derivative BTP2, an efficient inhibitor SOCE, identified that SOCE blockade suppresses proinflammatory gene expression. The impact of SOCE blockade on gene expression at the whole transcriptome level, however, is unknown. To fill this gap, we performed RNA sequencing (RNA-seq) and investigated at the whole transcriptome level the effect of BTP2 on gene expression in human peripheral blood mononuclear cells signaled with phytohemagglutinin. Our global gene expression analysis identified that SOCE blockade spares activation-induced expression of anti-inflammatory genes (e.g., IL10, TGFB1, FOXP3, and CTLA4) whereas the induced expression of proinflammatory genes such as IFNG and cytopathic genes such as GZMB are inhibited. We validated the differential expression of immunoregulatory genes identified by RNA-seq using preamplification-enhanced RT-qPCR assays. Because IL-2/IL2RA interaction is essential for T cell clonal expansion, we investigated and confirmed that BTP2 inhibits IL2RA expression at the protein level using multiparameter flow cytometry. Our elucidation that SOCE blockade spares activation-induced expression of anti-inflammatory genes while blocking pro-inflammatory gene expression suggests that SOCE blockers may represent a novel class of immunoregulatory drugs of value for treating autoimmune disease states and organ transplantation.

Discovery of selective Orai channel blockers bearing an indazole or a pyrazole scaffold.

The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 µM) and the nuclear translocation of NFAT at 30 and 10 µM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.

Sodium butyrate prevents cytokine-induced ß-cell dysfunction through restoration of stromal interaction molecule 1 expression and activation of store-operated calcium entry.

Sodium butyrate (NaB) improves ß-cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been fully elucidated. In this study, we investigated the impact of NaB on ß-cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 ß cells. Consistently, NaB improved glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the ß cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1ß-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent ß-cell death in response to IL-1ß treatment. Mechanistic experiments revealed that NaB mediated these beneficial effects in the ß-cell through histone deacetylase (HDAC) inhibition, iNOS suppression, and modulation of AKT-GSK-3 signaling. Taken together, these data support a model whereby NaB treatment promotes ß-cell function and Ca2+ homeostasis under proinflammatory conditions through pleiotropic effects that are linked with maintenance of SOCE. These results also suggest a relationship between ß-cell SOCE and gut microbiome-derived butyrate that may be relevant in the treatment and prevention of diabetes.

Septin regulation of Orai-mediated Ca2+ entry - a novel target for neurodegeneration.

Aberrant Ca2+ signaling is an early hallmark of multiple neurodegenerative syndromes including Alzheimer's and Parkinson's disease (AD and PD) as well as classes of rare genetic disorders such as Spinocebellar Ataxias. Therapeutic strategies that target aberrant Ca2+ signals whilst allowing normal neuronal Ca2+ signals have been a challenge. In a recent study Princen et al., performed a screen in the tauP301L cell model of AD for drugs that could specifically ameliorate the excess Ca2+ entry observed. They identified a class of compounds referred to as ReS19-T that interact with Septins, previously identified as regulators of the Store-operated Ca2+ entry channel Orai. Drug treatment of the cellular model, a mouse model and human iPSC derived neurons alleviate cellular and systemic deficits associated with tauP301L. Comparison of Septin filament architecture in disease conditions with and without the drug treatment indicate that excess Ca2+ entry is a consequence of abnormal Septin filament architecture resulting in aberrant ER-PM contacts. The importance of membrane contacts for maintaining precise cellular signaling has been recognized previously. However, the molecular mechanism by which Septin filaments organize the ER-PM junctions to regulate Ca2+ entry through Orai remains to be fully understood.

Novel insights into STIM1's role in store-operated calcium entry and its implications for T-cell mediated inflammation in trigeminal neuralgia.

This investigation aims to elucidate the novel role of Stromal Interaction Molecule 1 (STIM1) in modulating store-operated calcium entry (SOCE) and its subsequent impact on inflammatory cytokine release in T lymphocytes, thereby advancing our understanding of trigeminal neuralgia (TN) pathogenesis. Employing the Gene Expression Omnibus (GEO) database, we extracted microarray data pertinent to TN to identify differentially expressed genes (DEGs). A subsequent comparison with SOCE-related genes from the Genecards database helped pinpoint potential target genes. The STRING database facilitated protein-protein interaction (PPI) analysis to spotlight STIM1 as a gene of interest in TN. Through histological staining, transmission electron microscopy (TEM), and behavioral assessments, we probed STIM1's pathological effects on TN in rat models. Additionally, we examined STIM1's influence on the SOCE pathway in trigeminal ganglion cells using techniques like calcium content measurement, patch clamp electrophysiology, and STIM1- ORAI1 co-localization studies. Changes in the expression of inflammatory markers (TNF-α, IL-1ß, IL-6) in T cells were quantified using Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) in vitro, while immunohistochemistry and flow cytometry were applied in vivo to assess these cytokines and T cell count alterations. Our bioinformatic approach highlighted STIM1's significant overexpression in TN patients, underscoring its pivotal role in TN's etiology and progression. Experimental findings from both in vitro and in vivo studies corroborated STIM1's regulatory influence on the SOCE pathway. Furthermore, STIM1 was shown to mediate SOCE-induced inflammatory cytokine release in T lymphocytes, a critical factor in TN development. Supportive evidence from histological, ultrastructural, and behavioral analyses reinforced the link between STIM1-mediated SOCE and T lymphocyte-driven inflammation in TN pathogenesis. This study presents novel evidence that STIM1 is a key regulator of SOCE and inflammatory cytokine release in T lymphocytes, contributing significantly to the pathogenesis of trigeminal neuralgia. Our findings not only deepen the understanding of TN's molecular underpinnings but also potentially open new avenues for targeted therapeutic strategies.

Redox Enzymes P4HB and PDIA3 Interact with STIM1 to Fine-Tune Its Calcium Sensitivity and Activation.

Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.

Extended Synaptotagmins 1 and 2 Are Required for Store-Operated Calcium Entry, Cell Migration and Viability in Breast Cancer Cells.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1-STIM1 interaction at ER-PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

STIM1, ORAI1, and KDM2B in circulating tumor cells (CTCs) isolated from prostate cancer patients.

Introduction: Previous publications have shown that STIM1, ORAI1, and KDM2B, are implicated in Ca2+ signaling and are highly expressed in various cancer subtypes including prostate cancer. They play multiple roles in cancer cell migration, invasion, and metastasis. In the current study we investigated the expression of the above biomarkers in circulating tumor cells from patients with metastatic prostate cancer. Methods: Thirty-two patients were enrolled in this study and CTCs' isolation was performed with Ficoll density gradient. Two different triple immunofluorescence stainings were conducted with the following combination of antibodies: CK/KDM2B/CD45 and CK/STIM1/ORAI1. Slides were analyzed using VyCAP microscopy technology. Results: CTC-positive patients were detected in 41% for (CK/KDM2B/CD45) staining and in 56% for (CK/STIM1/ORAI1) staining. The (CK+/KDM2B+/CD45-) and the (CK+/STIM1+/ORAI1+) were the most frequent phenotypes as they were detected in 85% and 94% of the CTC-positive patients, respectively. Furthermore, the expression of ORAI1 and STIM1 in patients' PBMCs was very low exhibiting them as interesting specific biomarkers for CTC detection. The (CK+/STIM1+/ORAI1+) phenotype was correlated to bone metastasis (p = 0.034), while the (CK+/STIM1+/ORAI1-) to disease relapse (p = 0.049). Discussion: STIM1, ORAI1, and KDM2B were overexpressed in CTCs from patients with metastatic prostate cancer. STIM1 and ORAI1 expression was related to disease recurrence and bone metastasis. Further investigation of these biomarkers in a larger cohort of patients will clarify their clinical significance for prostate cancer patients.

Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Sodium Houttuyfonate Alleviates Monocrotaline-induced Pulmonary Hypertension by Regulating Orai1 and Orai2.

An increased intracellular Ca2+ concentration ([Ca2+]i) is a key trigger for pulmonary arterial smooth muscle cell (PASMC) proliferation and contributes greatly to pulmonary hypertension (PH). Extracellular Ca2+ influx via a store-operated Ca2+ channel, termed store-operated Ca2+ entry (SOCE), is a crucial mechanism for [Ca2+]i increase in PASMCs. Calcium release-activated calcium modulator (Orai) proteins, consisting of three members (Orai1-3), are the main components of the store-operated Ca2+ channel. Sodium houttuyfonate (SH) is a product of the addition reaction of sodium bisulfite and houttuynin and has antibacterial, antiinflammatory, and other properties. In this study, we assessed the contributions of Orai proteins to monocrotaline (MCT)-enhanced SOCE, [Ca2+]i, and cell proliferation in PASMCs and determined the effect of SH on MCT-PH and the underlying mechanism, focusing on Orai proteins, SOCE, and [Ca2+]i in PASMCs. Our results showed that: 1) Orai1 and Orai2 were selectively upregulated in the distal pulmonary arteries and the PASMCs of MCT-PH rats; 2) knockdown of Orai1 or Orai2 reduced SOCE, [Ca2+]i, and cell proliferation without affecting their expression in PASMCs in MCT-PH rats; 3) SH significantly normalized the characteristic parameters in a dose-dependent manner in the MCT-PH rat model; and 4) SH decreased MCT-enhanced SOCE, [Ca2+]i, and PASMC proliferation via Orai1 or Orai2. These results indicate that SH likely exerts its protective role in MCT-PH by inhibiting the Orai1,2-SOCE-[Ca2+]i signaling pathway.

Orai1/STIMs modulators in pulmonary vascular diseases.

Calcium (Ca2+) is a secondary messenger that regulates various cellular processes. However, Ca2+ mishandling could lead to pathological conditions. Orai1 is a Ca2+channel contributing to the store-operated calcium entry (SOCE) and plays a critical role in Ca2+ homeostasis in several cell types. Dysregulation of Orai1 contributed to severe combined immune deficiency syndrome, some cancers, pulmonary arterial hypertension (PAH), and other cardiorespiratory diseases. During its activation process, Orai1 is mainly regulated by stromal interacting molecule (STIM) proteins, especially STIM1; however, many other regulatory partners have also been recently described. Increasing knowledge about these regulatory partners provides a better view of the downstream signalling pathways of SOCE and offers an excellent opportunity to decipher Orai1 dysregulation in these diseases. These proteins participate in other cellular functions, making them attractive therapeutic targets. This review mainly focuses on Orai1 regulatory partners in the physiological and pathological conditions of the pulmonary circulation and inflammation.

The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry.

The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis.

Quercetin and Kaempferol inhibit HMC-1 activation via SOCE/NFATc2 signaling and suppress hippocampal mast cell activation in lipopolysaccharide-induced depressive mice.

OBJECTIVE AND DESIGN: Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin (Que) and kaempferol (Kae), as two major diet-derived flavonoids, inhibit MC activation and exhibit significant antidepressant effect due to their anti-inflammatory capacity. The study aimed to explore the mechanisms of inhibitory effect of Que and Kae on MC activation, and whether Que and Kae suppress hippocampal mast cell activation in LPS-induced depressive mice. SUBJECTS AND TREATMENT: In vitro assays, human mast cells (HMC-1) were pretreated with Que or Kae for 1 h, then stimulated by phorbol 12-myristate 13-acetate (PMA) and 2,5-di-t-butyl-1,4-benzohydroquinone (tBHQ) for 3 h or 12 h. In vivo assays, Que or Kae was administered by oral gavage once daily for 14 days and then lipopolysaccharide (LPS) intraperitoneally injection to induce depressive behaviors. METHODS: The secretion and expression of TNF-α were determined by ELISA and Western blotting. The nuclear factor of activated T cells (NFAT) transcriptional activity was measured in HMC-1 stably expressing NFAT luciferase reporter gene. Nuclear translocation of NFATc2 was detected by nuclear protein extraction and also was fluorescently detected in HMC-1 stably expressing eGFP-NFATc2. We used Ca2+ imaging to evaluate changes of store-operated calcium entry (SOCE) in HMC-1 stably expressing fluorescent Ca2+ indicator jGCamP7s. Molecular docking was used to assess interaction between the Que or Kae and calcium release-activated calcium modulator (ORAI). The  hippocampal mast cell accumulation and activation  were detected by toluidine blue staining and immunohistochemistry with ß-tryptase. RESULTS: In vitro assays of HMC-1 activated by PtBHQ (PMA and tBHQ), Que and Kae significantly decreased expression and secretion of TNF-α. Moreover, NFAT transcriptional activity and nuclear translocation of NFATc2 were remarkably inhibited by Que and Kae. In addition, the Ca2+ influx mediated by SOCE was suppressed by Que, Kae and the YM58483 (ORAI inhibitor), respectively. Importantly, the combination of YM58483 with Que or Kae had no additive effect on the inhibition of SOCE. The molecular docking also showed that Que and Kae both exhibit high binding affinities with ORAI at the same binding site as YM58483. In vivo assays, Que and Kae significantly reversed LPS-induced depression-like behaviors in mice, and inhibited hippocampal mast cell activation  in LPS-induced depressive mice. CONCLUSIONS: Our results indicated that suppression of SOCE/NFATc2 pathway-mediated by ORAI channels may be the mechanism of inhibitory effect of Que and Kae on MC activation, and also suggested Que and Kae may exert the antidepressant effect through suppressing hippocampal mast cell activation.

Vitamin K-dependent carboxylation in ß-cells and diabetes.

Vitamin K is an essential micronutrient and a cofactor for the enzyme γ-glutamyl carboxylase, which adds a carboxyl group to specific glutamic acid residues in proteins transiting through the secretory pathway. Higher vitamin K intake has been linked to a reduced incidence of type 2 diabetes (T2D) in humans. Preclinical work suggests that this effect depends on the γ-carboxylation of specific proteins in ß-cells, including endoplasmic reticulum Gla protein (ERGP), implicated in the control of intracellular Ca2+ levels. In this review we discuss these recent advances linking vitamin K and glucose metabolism, and argue that identification of γ-carboxylated proteins in ß-cells is pivotal to better understand how vitamin K protects from T2D and to design targeted therapies for this disease.