The effect of genetic polymorphisms in STIM1 and ORAI1 on erythropoietin resistance in Egyptian patients with end-stage renal disease.

Chronic renal failure (CRF) is an incurable disease with unique challenges. Anemia is a frequent complication affecting dialysis patients. Erythropoietin (EPO) is used to treat anemia, but a poor response may result. We investigated genetic polymorphisms of store-operated calcium channel (SOC) signaling, an important erythropoietin-activated pathway that may induce EPO resistance in patients with renal failure. A total of 108 end stage renal disease (ESRD) patients were selected for this study. Patients were divided into two groups according to their erythropoietin resistance index (ERI): 39 patients with an ERI>10 and 69 patients with an ERI<10. We selected four tagging single nucleotide polymorphisms (tSNPs) in STIM1 and five in ORAI1 in our study. A polymerase chain reaction was performed, and genotyping against EPO resistance was correlated. Patients with the AG genotype of rs1561876 in STIM1, the TC genotype of rs6486795 in ORAI1, and the TG or GG genotypes of rs12320939 in ORAI1 were associated with an increased risk of erythropoietin resistance. Overall, we reported a moderately significant relationship between genetic polymorphisms of STIM1 and EPO resistance. We also reported a highly significant relationship between genetic polymorphisms of ORAI1 and EPO resistance. The (A-A-G) haplotype of STIM1 and the (G-T-G-T-A, G-C-G-C-G, or G-T-T-C-G) haplotypes of ORAI1 were significantly associated with EPO resistance.

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.

Combined diosmin and bisoprolol attenuate cobalt chloride-induced cardiotoxicity and endothelial dysfunction through modulating miR-143-3P/MAPK/MCP-1, ERK5/CXCR4, Orai-1/STIM-1 signaling pathways.

Even while accelerated cardiomyocyte apoptosis is one of the primary causes of cardiac damage, the underlying mechanism is still mostly unknown. In addition to examining potential protective effects of bisoprolol and diosmin against CoCl2-induced cardiac injury, the goal of this study was to identify potential mechanisms regulating the hypoxic cardiac damage caused by cobalt chloride (CoCl2). For a period of 21 days except Cocl2 14 days from the first day of the experiment, rats were split into the following groups: Normal control group, rats received vehicle only (2 ml/kg/day, p.o.), (Cocl2, 150 mg/kg/day, p.o.), bisoprolol (25 mg/kg/day, p.o.); diosmin (100 mg/kg/day, p.o.) and bisoprolol + diosmin + Cocl2 groups. At the end of the experimental period, serum was taken for estimation of cardiac function, lipid profile, and pro/anti-inflammatory cytokines. Moreover, tissue samples were collected for evaluation of oxidative stress, endothelial dysfunction, α-SMA, PKC-α, MiR-143-3P, MAPK, ERK5, MCP-1, CXCR4, Orai-1, and STIM-1. Diosmin and bisoprolol, either alone or in combination, enhance heart function by reducing abnormalities in the electrocardiogram and the hypotension brought on by CoCl2. Additionally, they significantly ameliorate endothelial dysfunction by downregulating the cardiac expressions of α-SMA, PKC-α, MiR-143-3P, MAPK, ERK5, MCP-1, CXCR4, Orai-1, and STIM-1. Bisoprolol and diosmin produced modulatory activity against inflammatory state, redox balance, and atherogenic index concurrently. Together, diosmin and bisoprolol, either alone or in combination, significantly reduced all the cardiac alterations brought on by CoCl2. The capacity to obstruct hypoxia-induced α-SMA, PKC-α, MiR-143-3P/MAPK/MCP-1, MiR-143-3P/ERK5/CXCR4, Orai-1/STIM-1 signaling activation, as well as their anti-inflammatory, antioxidant, and anti-apoptotic properties, may be responsible for these cardio-protective results.

ELD607 specifically traffics Orai1 to the lysosome leading to inhibition of store operated calcium entry.

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer.

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.

Orai1 and Orai3 act through distinct signalling axes to promote stemness and tumorigenicity of breast cancer stem cells.

BACKGROUND: One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. METHODS: A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. RESULTS: Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. CONCLUSION: We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

L-type Ca2+ channel activation of STIM1-Orai1 signaling remodels the dendritic spine ER to maintain long-term structural plasticity.

Learning and memory require coordinated structural and functional plasticity at neuronal glutamatergic synapses located on dendritic spines. Here, we investigated how the endoplasmic reticulum (ER) controls postsynaptic Ca2+ signaling and long-term potentiation of dendritic spine size, i.e., sLTP that accompanies functional strengthening of glutamatergic synaptic transmission. In most ER-containing (ER+) spines, high-frequency optical glutamate uncaging (HFGU) induced long-lasting sLTP that was accompanied by a persistent increase in spine ER content downstream of a signaling cascade engaged by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter being activated by stromal interaction molecule 1 (STIM1) in response to ER Ca2+ release. In contrast, HFGU stimulation of ER-lacking (ER-) spines expressed only transient sLTP and exhibited weaker Ca2+ signals noticeably lacking Orai1 and ER contributions. Consistent with spine ER regulating structural metaplasticity, delivery of a second stimulus to ER- spines induced ER recruitment along with persistent sLTP, whereas ER+ spines showed no additional increases in size or ER content in response to sequential stimulation. Surprisingly, the physical interaction between STIM1 and Orai1 induced by ER Ca2+ release, but not the resulting Ca2+ entry through Orai1 channels, proved necessary for the persistent increases in both spine size and ER content required for expression of long-lasting late sLTP.

Targeting STIM1 Contributes to Alleviating Remodeling of Vascular Smooth Muscle Cells in Atherosclerosis.

BACKGROUND: Remodeling of vascular smooth muscle cells (VSMCs), as a pathological hallmark of cardiovascular diseases, is related to the molecular rewiring of Calcium signaling, which induces upregulation of stromal interaction molecule (STIM) proteins. This study analyzed the influence of STIM1 proteins on the remodeling of VSMCs in atherosclerosis (AS). METHODS: After oxidized low-density lipoprotein (ox-LDL) treatment and transfection, VSMC viability, migration, and invasion were separately measured using Cell Counting Kit-8, Scratch assay, and Transwell assay. An animal AS model was constructed, and histological analysis via hematoxylin-eosin staining was conducted on the aorta. RESULTS: Ox-LDL promoted expression of STIM1 and Orai calcium release-activated calcium modulator 1 (Orai1). STIM1 or Orai1 downregulation suppressed viability, migration, invasion, and phenotypic switching of ox-LDL-treated VSMCs, whereas STIM1 or Orai1 upregulation had opposite effects. Orai1 level was upregulated by STIM1 overexpression. Orai1 silencing reversed the effects of STIM1 overexpression in VSMCs. STIM1 deficiency alleviated AS and regulated expression of Orai1 and phenotypic switch-related factors in vivo. CONCLUSION: STIM1 deficiency suppresses viability, migration, invasion, and phenotypic switching of ox-LDL-induced VSMCs and alleviates AS by inhibiting Orai1.

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.

Targeting calciumopathy for neuroprotection: focus on calcium channels Cav1, Orai1 and P2X7.

As the uncontrolled entry of calcium ions (Ca2+) through plasmalemmal calcium channels is a cell death trigger, the conjecture is here raised that mitigating such an excess of Ca2+ entry should rescue from death the vulnerable neurons in neurodegenerative diseases (NDDs). However, this supposition has failed in some clinical trials (CTs). Thus, a recent CT tested whether isradipine, a blocker of the Cav1 subtype of voltage-operated calcium channels (VOCCs), exerted a benefit in patients with Parkinson's disease (PD); however, outcomes were negative. This is one more of the hundreds of CTs done under the principle of one-drug-one-target, that have failed in Alzheimer's disease (AD) and other NDDs during the last three decades. As there are myriad calcium channels to let Ca2+ ions gain the cell cytosol, it seems reasonable to predict that blockade of Ca2+ entry through a single channel may not be capable of preventing the Ca2+ flood of cells by the uncontrolled Ca2+ entry. Furthermore, as Ca2+ signaling is involved in the regulation of myriad functions in different cell types, it seems also reasonable to guess that a therapy should be more efficient by targeting different cells with various drugs. Here, we propose to mitigate Ca2+ entry by the simultaneous partial blockade of three quite different subtypes of plasmalemmal calcium channels that is, the Cav1 subtype of VOCCs, the Orai1 store-operated calcium channel (SOCC), and the purinergic P2X7 calcium channel. All three channels are expressed in both microglia and neurons. Thus, by targeting the three channels with a combination of three drug blockers we expect favorable changes in some of the pathogenic features of NDDs, namely (i) to mitigate Ca2+ entry into microglia; (ii) to decrease the Ca2+-dependent microglia activation; (iii) to decrease the sustained neuroinflammation; (iv) to decrease the uncontrolled Ca2+ entry into neurons; (v) to rescue vulnerable neurons from death; and (vi) to delay disease progression. In this review we discuss the arguments underlying our triad hypothesis in the sense that the combination of three repositioned medicines targeting Cav1, Orai1, and P2X7 calcium channels could boost neuroprotection and delay the progression of AD and other NDDs.

Store-operated calcium entry dysfunction in CRAC channelopathy: Insights from a novel STIM1 mutation.

Store-operated calcium entry (SOCE) plays a crucial role in maintaining cellular calcium homeostasis. This mechanism involves proteins, such as stromal interaction molecule 1 (STIM1) and ORAI1. Mutations in the genes encoding these proteins, especially STIM1, can lead to various diseases, including CRAC channelopathies associated with severe combined immunodeficiency. Herein, we describe a novel homozygous mutation, NM_003156 c.792-3C > G, in STIM1 in a patient with a clinical profile of CRAC channelopathy, including immune system deficiencies and muscle weakness. Functional analyses revealed three distinct spliced forms in the patient cells: wild-type, exon 7 skipping, and intronic retention. Calcium influx analysis revealed impaired SOCE in the patient cells, indicating a loss of STIM1 function. We developed an antisense oligonucleotide treatment that improves STIM1 splicing and highlighted its potential as a therapeutic approach. Our findings provide insights into the complex effects of STIM1 mutations and shed light on the multifaceted clinical presentation of the patient.

Association of HLA class II gene polymorphisms and expression levels of ORAI1/STIM1 genes in HIV-1‒positive patients with HIV-related dermatoses in Latvia.

INTRODUCTION: This study explores the immunogenetic associations of human leukocyte antigens (HLA) and the calcium release-activated calcium modulator 1 (ORAI1) and stromal interaction molecule 1 (STIM1) genes in HIV-1‒positive patients with HIV-related skin disorders. METHODS: This study assessed the distribution of variants of HLA class II alleles and expression levels of ORAI1 and STIM1 genes in the blood between HIV-1‒positive patients with HIV-related skin disorders and the control group with no HIV within the Latvian population. RESULTS: The research group comprised 115 HIV-1‒positive patients with HIV-related skin disorders, and the control group included 80 healthy individuals. Risk alleles (HLA- DQB1*02:01-0301 and HLA-DQA1*01:01-0501) and protective alleles (HLA-DRB1*07-13, DRB1*01-13, DRB1*04-11, and HLA-DQA1*05:01-0501) showed statistical significance in the groups. In 38 out of 115 patients, higher expression levels of ORAI1 and STIM1 genes were detected in the blood at the beginning of treatment. A significantly higher level of the microribonucleic acid (mRNA) ORAI1 gene was also found in the control group. CONCLUSIONS: The results demonstrate that HLA class II alleles are associated with a trend toward risk/protection concerning HIV-related skin disorders in HIV-1‒positive patients. It was also shown that a low level of ORAI1 mRNA and the risk allele HLA-DQB1*0201-0301 were simultaneously present in the research group.

IL-37 protects against house dust mite-induced airway inflammation and airway epithelial barrier dysfunction via inhibiting store-operated calcium entry.

BACKGROUND: Airway epithelial barrier dysfunction has been proved to contribute to the development of type 2 inflammation of asthma. Interleukin (IL)-37 is a negative regulator of immune responses and allergic airway inflammation. However, whether IL-37 has any effect on airway epithelial barrier has been unknown. METHODS: We evaluated the role of IL-37 in both mouse model and cultured 16HBE cells. Histology and ELISA assays were used to evaluate airway inflammation. FITC-dextran permeability assay was used to evaluate the airway epithelial barrier function. Immunofluorescence, western blot and quantitative Real-Time PCR (RT-PCR) were used to evaluate the distribution and expression of tight junction proteins. RT-PCR and Ca2+ fluorescence measurement were used to evaluate the mRNA expression and activity of store-operated calcium entry (SOCE). RESULTS: IL-37 inhibited house dust mite (HDM)-induced airway inflammation and decreased the levels of IgE in serum and type 2 cytokines in bronchoalveolar lavage fluid (BALF) compared to asthmatic mice. IL-37 protected against HDM-induced airway epithelial barrier dysfunction, including reduced leakage of FITC-dextran, enhanced expression of TJ proteins, and restored the membrane distribution of TJ proteins. Moreover, IL-37 decreased the level of IL-33 in the BALF of asthmatic mice and the supernatants of HDM-treated 16HBE cells. IL-37 decreased the peak level of Ca2+ fluorescence induced by thapsigargin and HDM, and inhibited the mRNA expression of Orai1, suggesting an inhibiting effect of IL-37 on SOCE in airway epithelial cells. CONCLUSION: IL-37 plays a protective role in airway inflammation and HDM-induced airway epithelial barrier dysfunction by inhibiting SOCE.

STIM1 mediates methamphetamine-induced neuronal autophagy and apoptosis.

Methamphetamine (METH) is a widely abused amphetamine-type psychoactive drug that causes serious health problems. Previous studies have demonstrated that METH can induce neuron autophagy and apoptosis in vivo and in vitro. However, the molecular mechanisms underlying METH-induced neuron autophagy and apoptosis remain poorly understood. Stromal interacting molecule 1 (STIM1) was hypothesized to be involved in METH-induced neuron autophagy and apoptosis. Therefore, the expression of STIM1 protein was measured and the effect of blocking STIM1 expression with siRNA was investigated in cultured neuronal cells, and the hippocampus and striatum of mice exposed to METH. Furthermore, intracellular calcium concentration and endoplasmic reticulum (ER) stress-related proteins were determined in vitro and in vivo in cells treated with METH. The results suggested that STIM1 mediates METH-induced neuron autophagy by activating the p-Akt/p-mTOR pathway. METH exposure also resulted in increased expression of Orai1, which was reversed after STIM1 silencing. Moreover, the disruption of intracellular calcium homeostasis induced ER stress and up-regulated the expression of pro-apoptotic protein CCAAT/enhancer-binding protein homologous protein (CHOP), resulting in classic mitochondria apoptosis. METH exposure can cause neuronal autophagy and apoptosis by increasing the expression of STIM1 protein; thus, STIM1 may be a potential gene target for therapeutics in METH-caused neurotoxicity.

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.

Role of STIM1 in stretch-induced signaling in human airway smooth muscle.

Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1, and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways, including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.NEW & NOTEWORTHY Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.

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.

Deletion of myeloid-specific Orai1 calcium channel does not affect pancreatic tissue damage in experimental acute pancreatitis.

BACKGROUND: Store-operated Ca2+ entry (SOCE) mediated by ORAI1 channel plays a crucial role in acute pancreatitis (AP). Macrophage is an important regulator in amplifying pancreatic tissue damage, but little is known about the role of ORAI1 in macrophages. In this study, we examined the effects of macrophage-specific ORAI1 on pancreatic tissue damage in AP. METHOD: Myeloid-specific Orai1 deficient mice was generated by crossing a LysM-Cre mouse line with Orai1f/f mice. Bone marrow-derived macrophages (BMDMs) were isolated, cultured, and stimulated to induce M1 or M2 macrophage polarization. Intracellular Ca2+ signals were measured by time-lapse confocal microscope imaging, with a Ca2+ indicator (Fluo 4). Experimental AP was induced by hourly intraperitoneal injections of caerulein or retrograde biliopancreatic infusion of sodium taurocholate. Pancreatic tissue damage was assessed by histopathological scoring and immunostaining. Sepsis was induced by intraperitoneal injection of lipopolysaccharide; organ damage and serum pro-inflammatory cytokines were measured. RESULT: Myeloid-specific Orai1 deletion exhibited minimal effect on SOCE in M0 macrophages and promoted M2 macrophage polarization ex vivo. Myeloid-specific Orai1 deletion did not affect pancreatic tissue damage, nor neutrophil or macrophage infiltration in two models of AP. Similarly, myeloid-specific Orai1 deletion did not influence overall survival rate in a model of sepsis, nor lung, kidney, and liver damage; while serum pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1ß were higher in Orai1ΔLysM mice, but were largely reduced in mice with Orai1 inhibitor. CONCLUSION: Our data suggest that ORAI1 may not be a predominant SOCE channel in macrophages and play a limited role in mediating pancreatic tissue damage in AP.