Store-operated Calcium Research Articles

1,2,3,4-dithiadiazole derivatives as a novel class of calcium signaling modulators

Aberrant calcium signaling is associated with a diverse range of pathologies, including cardiovascular and neurodegenerative diseases, diabetes, cancer, etc… So, therapeutic strategies based on the correction of pathological calcium signaling are becoming extremely in demand. Thus, the development of novel calcium signaling modulators remains highly actual. Previously we found that 1,2,3,4-dithiadiazole derivative 3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxide can strongly reduce calcium uptake through store-operated calcium (SOC) channels. Here we tested several structurally related compounds and found that most of them can effectively affect SOC channels and attenuate calcium content in the endoplasmic reticulum, thus, establishing 1,2,3,4-dithiadiazoles as a novel class of SOC channel inhibitors. Comparing different 1,2,3,4-dithiadiazole derivatives we showed that previously published 3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxide and newly tested 3-(3,5-difluorophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole 2-oxide demonstrated the highest efficacy of SOC entry reduction, supposing the important role of electron-withdrawing substituents to realize the inhibitory activity of 1,2,3,4-dithiadiazoles.

Robust expression of the TRPC1 channel associated with photoreceptor loss in the rat retina

Baseline intracellular calcium levels are significantly higher in neuronal and glial cells of rat retinas with retinitis pigmentosa (RP). Although this situation could initiate multiple detrimental pathways that lead to cell death, we considered the possibility of TRPC1 being involved in maintaining calcium homeostasis in the retina by acting as a component of store-operated calcium (SOC) channels with special relevance during photoreceptor degeneration. In this study, we examined by Western blot the expression of TRPC1 in healthy control rat retinas (Sprague-Dawley, SD) and retinas with RP (P23H-1 rats). We also analyzed its specific cellular distribution by immunofluorescence to recognize changes during neurodegeneration and to determine whether its presence is consistent with high basal calcium levels and cellular survival in degenerating retinas. We found that TRPC1 immunostaining was widely distributed across the retina in both rat strains, SD and P23H, and its expression levels significantly increased in the retinas with advanced degeneration compared to the age-control SD rats. In the outer retina, TRPC1 immunoreactivity was distributed in pigment epithelium cells, the photoreceptor inner segments of older animals, and the outer plexiform layer. In the inner retina, TRPC1 labeling was detected in horizontal cells, specific somata of bipolar and amacrine cells, and cellular processes in all the strata of the inner plexiform layer. Somata and processes were also highly immunoreactive in the ganglion cell layer and astrocytes in the nerve fiber layer in all animals. In the P23H rat retinas, the TRPC1 distribution pattern changed according to advancing photoreceptor degeneration and the gliosis reaction, with TRPC1 immunoreactive Müller cells mainly in advanced stages of disease. The cellular TRPC1 immunoreactivity found in this work suggests different mechanisms of activation of these channels depending on the cell type. Furthermore, the results support the idea that photoreceptor loss due to RP is associated with robust TRPC1 protein expression in the rat inner retina and raise the possibility of TRPC1 channels contributing to maintain high basal calcium levels during neurodegeneration and/or maintenance processes of the inner retina.

Protease-activated receptor-1 mediated calcium oscillations in renal mesangial cells

INTRODUCTION. Mesangial cells provide structural support to the glomerular tuft and modulate the glomerular capillary flow via their contractile properties. Mesangial cells undergo phenotypic changes into myofibroblast-like cells, such as proliferation, mesangial expansion, abnormal glomerular tuft formation, and reduced numbers of capillary loops, are present in several glomerular diseases, including diabetic nephropathy and glomerulonephritis. In addition, thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling and mechanisms in mesangial cells were not examined. This study aims to investigate protease-activated mechanisms regulating mesangial cell contraction and glomerular capillary flow. METHODS. We used primary human renal mesangial cells (HRMC) to determine the signaling mechanisms mediated by PAR1 thrombin-activated receptors. Confocal fluorescent microscopy was utilized to detect changes in intracellular Ca 2+ response to specific PAR1 modulators. Pharmacology and patch clamp electrophysiology was further applied to reveal downstream signaling mechanisms responsible for intracellular Ca 2+ oscillations. RESULTS. PAR1-mediated Ca 2+ response displayed high sensitivity to specific agonist (TFLLR-NH 2 ) with EC 50 values of 3 and 6.3 nM for male and female-derived cultured cells, respectively (the competition of a ligand for receptor binding fit converged; adj. R 2 =0.98). The response to PAR1 activation promoted initial cytosolic Ca 2+ increase followed by synchronized, damped Ca 2+ oscillations with a lag of 6.74±0.84 min between peaks. The pre-application of a specific inhibitor (RWJ56110) eliminated PAR1-mediated response, and oscillations were blocked by the changes of an extracellular solution to zero Ca 2+ . The specific inhibitors for store-operated calcium (SOCs) (Pyr6) and TRPC3 (GSK 2833503A) channels strongly attenuated oscillation behavior (up to 40% when added separately and up to 65% when both were applied; two-way ANOVA, * p<0.0001). In addition, the effect of a specific inhibitor of TRPC6 channels (BI-749327) had a minimal impact on Ca 2+ flux. Further single-channel electrophysiology experiments in HRMC cells confirmed the involvement of SOC and TRPC3 channels in PAR1-mediated GPCR activation. CONCLUSION. Our results indicate that coagulation proteases like thrombin may strongly regulate mesangial cell contraction and corresponding glomerular capillary flow by PAR1 GPCRs-related activation. The contraction mechanism is mediated presumably through SOCs entry and TRPC3 channels. Since high thrombin levels are linked to poor diabetic control, the described signaling may play a crucial role in the development of diabetic glomerular disease. DK126720 (to OP), DK129227 (to AS and OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The pair ceramide 1-phosphate/ceramide kinase regulates intracellular calcium and progesterone-induced human sperm acrosomal exocytosis.

Before fertilization, spermatozoa must undergo calcium-regulated acrosome exocytosis in response to physiological stimuli such as progesterone and zona pellucida. Our laboratory has elucidated the signaling cascades accomplished by different sphingolipids during human sperm acrosomal exocytosis. Recently, we established that ceramide increases intracellular calcium by activating various channels and stimulating the acrosome reaction. However, whether ceramide induces exocytosis on its own, activation of the ceramide kinase/ceramide 1-phosphate (CERK/C1P) pathway or both is still an unsolved issue. Here, we demonstrate that C1P addition induces exocytosis in intact, capacitated human sperm. Real-time imaging in single-cell and calcium measurements in sperm population showed that C1P needs extracellular calcium to induce [Ca2+]i increase. The sphingolipid triggered the cation influx through voltage-operated calcium (VOC) and store-operated calcium (SOC) channels. However, it requires calcium efflux from internal stores through inositol 3-phosphate receptors (IP3R) and ryanodine receptors (RyR) to achieve calcium rise and the acrosome reaction. We report the presence of the CERK in human spermatozoa, the enzyme that catalyzes C1P synthesis. Furthermore, CERK exhibited calcium-stimulated enzymatic activity during the acrosome reaction. Exocytosis assays using a CERK inhibitor demonstrated that ceramide induces acrosomal exocytosis, mainly due to C1P synthesis. Strikingly, progesterone required CERK activity to induce intracellular calcium increase and acrosome exocytosis. This is the first report, implicating the bioactive sphingolipid C1P in the physiological progesterone pathway leading to the sperm acrosome reaction.

The STIM1/2-Regulated Calcium Homeostasis Is Impaired in Hippocampal Neurons of the 5xFAD Mouse Model of Alzheimer’s Disease

Alzheimer's disease (AD) is the most common cause of age-related dementia. Neuronal calcium homeostasis impairment may contribute to AD. Here we demonstrated that voltage-gated calcium (VGC) entry and store-operated calcium (SOC) entry regulated by calcium sensors of intracellular calcium stores STIM proteins are affected in hippocampal neurons of the 5xFAD transgenic mouse model. We observed excessive SOC entry in 5xFAD mouse neurons, mediated by STIM1 and STIM2 proteins with increased STIM1 contribution. There were no significant changes in cytoplasmic calcium level, endoplasmic reticulum (ER) bulk calcium levels, or expression levels of STIM1 or STIM2 proteins. The potent inhibitor BTP-2 and the FDA-approved drug leflunomide reduced SOC entry in 5xFAD neurons. In turn, excessive voltage-gated calcium entry was sensitive to the inhibitor of L-type calcium channels nifedipine but not to the T-type channels inhibitor ML218. Interestingly, the depolarization-induced calcium entry mediated by VGC channels in 5xFAD neurons was dependent on STIM2 but not STIM1 protein in cells with replete Ca2+ stores. The result gives new evidence on the VGC channel modulation by STIM2. Overall, the data demonstrate the changes in calcium signaling of hippocampal neurons of the AD mouse model, which precede amyloid plaque accumulation or other signs of pathology manifestation.

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid.

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.

ORAI2 Promotes Gastric Cancer Tumorigenicity and Metastasis through PI3K/Akt Signaling and MAPK-Dependent Focal Adhesion Disassembly.

The ubiquitous second messenger Ca2+ has long been recognized as a key regulator in cell migration. Locally confined Ca2+, in particular, is essential for building front-to-rear Ca2+ gradient, which serves to maintain the morphologic polarity required in directionally migrating cells. However, little is known about the source of the Ca2+ and the mechanism by which they crosstalk between different signaling pathways in cancer cells. Here, we report that calcium release-activated calcium modulator 2 (ORAI2), a poorly characterized store-operated calcium (SOC) channel subunit, predominantly upregulated in the lymph node metastasis of gastric cancer, supports cell proliferation and migration. Clinical data reveal that a high frequency of ORAI2-positive cells in gastric cancer tissues significantly correlated with poor differentiation, invasion, lymph node metastasis, and worse prognosis. Gain- and loss-of-function showed that ORAI2 promotes cell motility, tumor formation, and metastasis in both gastric cancer cell lines and mice. Mechanistically, ORAI2 mediated SOC activity and regulated tumorigenic properties through the activation of the PI3K/Akt signaling pathways. Moreover, ORAI2 enhanced the metastatic ability of gastric cancer cells by inducing FAK-mediated MAPK/ERK activation and promoted focal adhesion disassembly at rear-edge of the cell. Collectively, our results demonstrate that ORAI2 is a novel gene that plays an important role in the tumorigenicity and metastasis of gastric cancer. SIGNIFICANCE: These findings describe the critical role of ORAI2 in gastric cancer cell migration and tumor metastasis and uncover the translational potential to advance drug discovery along the ORAI2 signaling pathway.

Germline Genetic Association between Stromal Interaction Molecule 1 (STIM1) and Clinical Outcomes in Breast Cancer Patients.

Among all cancers in women, breast cancer has the highest incidence. The mortality of breast cancer is highly associated with metastasis. Migration and malignant transformation of cancer cells have been reported to be modulated by store-operated calcium (SOC) channels, which control calcium signaling and cell proliferation pathways. Stromal interaction molecule 1 (STIM1) is a calcium sensor in the endoplasmic reticulum, triggering the activation of store-operated calcium signaling. However, the clinical relevance of STIM1 in breast cancer is still unclear. Here, we recruited 348 breast cancer patients and conducted a genetic association study to address this question. Four tagging germline single nucleotide variants (SNVs) in STIM1 were selected and RNA sequencing data of 525 breast cancer samples from The Cancer Genome Atlas (TCGA) database were evaluated. The results show that rs2304891 and rs3750996 were correlated with clinical stage of breast cancer. Expression quantitative trait loci (eQTL) analysis indicated that risk G allele of STIM1 contributed to the higher expression of STIM1. In addition, we found an increased risk of rs2304891 G allele and rs3750996 A allele in estrogen receptor (ER) positive and progesterone receptor (PR) positive patients. In conclusion, our results suggest that germline SNV, rs2304891 and rs3750996 as well as STIM1 expression are important biomarkers for the prediction of clinical outcomes in breast cancer patients.

Evaluation for the Genetic Association between Store-Operated Calcium Influx Pathway (STIM1 and ORAI1) and Human Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Infection.

Simple SummaryIn this study, we systematically evaluated the genetic susceptibility between the store-operated calcium (SOC) influx pathway (stromal interaction molecule 1 (STIM1) and ORAI1) and human hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB) infection. In total, 3631 patients with CHB were recruited, forty polymorphisms of STIM1 and ORAI1 were comprehensively analyzed. Three single-nucleotide polymorphisms (SNPs) of STIM1 (rs6578418, rs11030472, and rs7116520) and one SNP of ORAI1 (rs6486795) showed a trend of being significantly associated with HCC. In particular, our functional studies (images from total internal reflection fluorescence microscopy and transwell migration assay) revealed that calcium (Ca2+) signaling is essential for the migration of HCC. Based on such a comprehensively screening in 3631 patients with chronic hepatitis, our results indicate the important role of store-operated calcium pathways in HCC.Hepatocellular carcinoma (HCC) often develops from chronic hepatitis B (CHB) through replication of hepatitis B virus (HBV) infection. Calcium (Ca2+) signaling plays an essential role in HBV replication. Store-operated calcium (SOC) channels are a major pathway of Ca2+ entry into non-excitable cells such as immune cells and cancer cells. The basic components of SOC signaling include the STIM1 and ORAI1 genes. However, the roles of STIM1 and ORAI1 in HBV-mediated HCC are still unclear. Thus, long-term follow-up of HBV cohort was carried out in this study. This study recruited 3631 patients with chronic hepatitis (345 patients with HCC, 3286 patients without HCC) in a Taiwanese population. Genetic variants of the STIM1 and ORAI1 genes were detected using an Axiom CHB1 genome-wide array. Clinical associations of 40 polymorphisms were analyzed. Three of the STIM1 single-nucleotide polymorphisms (SNPs) (rs6578418, rs7116520, and rs11030472) and one SNP of ORAI1 (rs6486795) showed a trend of being associated with HCC disease (p < 0.05). However, after correction for multiple testing, none of the SNPs reached a significant level (q > 0.05); in contrast, neither STIM1 nor ORAI1 showed a significant association with HCC progression in CHB patients. Functional studies by both total internal reflection fluorescence images and transwell migration assay indicated the critical roles of SOC-mediated signaling in HCC migration. In conclusion, we reported a weak correlation between STIM1/ORAI1 polymorphisms and the risk of HCC progression in CHB patients.

LPS-induced vein endothelial cell injury and acute lung injury have Btk and Orai 1 to regulate SOC-mediated calcium influx

Patients with sepsis and sepsis-related complications have a high mortality. Endothelial cell dysfunction plays a central role in sepsis pathophysiological process. In sepsis patients, endothelial cell apoptosis is associated with intracellular calcium overload. Multiple functions in the apoptotic process have been found to be regulated by calcium signaling. Our previous work had proved that LPS-induced cell injury was associated with store-operated calcium (SOC) entry mediated by stromal interaction molecule-1 (STIM 1) in Human umbilical vein endothelial cells (HUVEC), but the underlying molecular mechanism has not been adequately defined. Here we report that the LPS-induced cell injury is related to the calcium overload in HUVEC. SOC entry mediated by calcium release-activated calcium modulator (Orai) 1 and transient receptor potential canonical (TRPC) 1 was associated with LPS-induced calcium overload and cell apoptosis. Bruton’s tyrosine kinase (Btk)/Phospholipase C(PLC) γ/inositol 1,4,5-triphosphate receptor (IP3R) played a major role in regulating calcium overload in LPS-induced HUVEC. Knockdown of Btk markedly inhibited the expressions of Orai 1 and its downstream molecule IP3R but not that of TRPC1 in LPS-induced HUVEC. In mice, knockdown of Btk and Orai 1 inhibited LPS-induced calcium overload, pulmonary vascular endothelial cell (VEC) injury and acute lung injury. These findings demonstrated that Btk acts as a regulator of calcium-dependent signaling, especially in the Orai 1-mediated SOC entry of the LPS-induced VEC.

Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle

Background and purposeMetabolic syndrome causes adverse effects on the coronary circulation including altered vascular responsiveness and the progression of coronary artery disease (CAD). However the underlying mechanisms linking obesity with CAD are intricated. Augmented vasoconstriction, mainly due to impaired Ca2+ homeostasis in coronary vascular smooth muscle (VSM), is a critical factor for CAD. Increased calcium–induced calcium release (CICR) mechanism has been associated to pathophysiological conditions presenting persistent vasoconstriction while increased store operated calcium (SOC) entry appears to activate proliferation and migration in coronary vascular smooth muscle (VSM). We analyze here whether metabolic syndrome might alter SOC entry as well as CICR mechanism in coronary arteries, contributing thus to a defective Ca2+ handling and therefore accelerating the progression of CAD. Experimental approachMeasurements of intracellular Ca2+ ([Ca2+]i) and tension and of Ca2+ channels protein expression were performed in coronary arteries (CA) from lean Zucker rats (LZR) and obese Zucker rats (OZR). Key resultsSOC entry stimulated by emptying sarcoplasmic reticulum (SR) Ca2+ store with cyclopiazonic acid (CPA) was decreased and associated to decreased STIM-1 and Orai1 protein expression in OZR CA. Further, CICR mechanism was blunted in these arteries but Ca2+ entry through voltage-dependent L-type channels was preserved contributing to maintain depolarization-induced increases in [Ca2+]i and vasoconstriction in OZR CA. These results were associated to increased expression of voltage-operated L-type Ca2+ channel alpha 1C subunit (CaV1.2) but unaltered ryanodine receptor (RyR) and sarcoendoplasmic reticulum Ca2+‐ATPase (SERCA) pump protein content in OZR CA. Conclusion and implicationsThe present manuscript provides evidence of impaired Ca2+ handling mechanisms in coronary arteries in metabolic syndrome where a decrease in both SOC entry and CICR mechanism but preserved vasoconstriction are reported in coronary arteries from obese Zucker rats. Remarkably, OZR CA VSM at this state of metabolic syndrome seemed to have developed a compensation mechanism for impaired CICR by overexpressing CaV1.2 channels.

Abnormal Ca2+ handling contributes to the impairment of aortic smooth muscle contractility in Zucker diabetic fatty rats

Vascular dysfunction is a common pathological basis for complications in individuals affected by diabetes. Previous studies have established that endothelial dysfunction is the primary contributor to vascular complications in type 2 diabetes (T2DM). However, the role of vascular smooth muscle cells (VSMCs) in vascular complications associated with T2DM is still not completely understood. The aim of this study is to explore the potential mechanisms associated with Ca2+ handling dysfunction and how this dysfunction contributes to diabetic vascular smooth muscle impairment. The results indicated that endothelium-dependent vasodilation was impaired in diabetic aortae, but endothelium-independent vasodilation was not altered. Various vasoconstrictors such as phenylephrine, U46619 and 5-HT could induce vasoconstriction in a concentration-dependent manner, such that the dose-response curve was parallel shifted to the right in diabetic aortae, compared to the control. Vasoconstrictions mediated by L-type calcium (Cav1.2) channels were attenuated in diabetic aortae, but effects mediated by store-operated calcium (SOC) channels were enhanced. Intracellular Ca2+ concentration ([Ca2+]i) in VSMCs was detected by Fluo-4 calcium fluorescent probes, and demonstrated that SOC-mediated Ca2+ entry was increased in diabetic VSMCs. VSMC-specific knockout of STIM1 genes decreased SOC-mediated and phenylephrine-induced vasoconstrictive response in mice aortae. Additionally, Orai1 expression was up-regulated, Cav1.2 expression was downregulated, and the phenotypic transformation of diabetic VSMCs was determined in diabetic aortae. The overexpression of Orai1 markedly promoted the OPN expression of VSMCs, whereas SKF96365 (SOC channel blocker) reversed the phenotypic transformation of diabetic VSMCs. Our results demonstrated that the vasoconstriction response of aortic smooth muscle was weakened in type 2 diabetic rats, which was related to the downregulation of the Cav1.2 channel and the up-regulation of the SOC channel signaling pathway.

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels.We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms

AbstractApproximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate–dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.

Orai1: CRACing the Th17 response in AKI.

A strong Th17 inflammatory response aggravates ischemia reperfusion-induced (IR-induced) acute kidney injury (AKI), tissue fibrosis, and AKI-to-chronic kidney disease (CKD) progression. However, the underlying mechanisms of sustained Th17 activation following AKI and during AKI-to-CKD progression are unclear. In this issue of the JCI, Mehrotra et al. present compelling evidence that the store-operated calcium (Ca2+) channel Orai1 sustains Th17-driven inflammatory response after AKI and drives the AKI-to-CKD transition. Orai1 blockade significantly protected renal function from IR, attenuated high-salt-induced AKI-to-CKD progression in rats, and decreased Th17 response in rat and human T cells. Therapeutic targeting of Orai1 can potentially reduce AKI, AKI-to-CKD progression, and other Th17-driven diseases.

Comparison of Ca2+ Handling for the Regulation of Vasoconstriction between Rat Coronary and Renal Arteries

Background: Ca²⁺ plays an important role in the regulation of vasoconstriction. Ca²⁺ signaling is regulated by a number of Ca²⁺-handling proteins. However, whether differences in Ca²⁺ handling affect the regulation of vasoconstriction in different arteries remains elusive. Objective: To determine whether differences in Ca²⁺ handling affect the response to vasoconstrictors in different arteries. Methods: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca²⁺ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. Results: Phenylephrine did not induce ­coronary arterial contraction. There were differences in ­5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F_2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca²⁺ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. Conclusions: Ca²⁺ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.

Peripheral myelin protein 22 modulates store-operated calcium channel activity, providing insights into Charcot-Marie-Tooth disease etiology

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400–403), which is located within the CAD/SOAR domain. We determined this segment’s specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

Calcium Imaging of Store-Operated Calcium (Ca2+) Entry (SOCE) in HEK293 Cells Using Fura-2.

The store-operated calcium (Ca2+) entry (SOCE) pathway is an essential Ca2+ signaling pathway in non-excitable cells that serve many physiological functions. SOCE is mediated through the plasma membrane (PM) protein, Orai1, and the endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1). One of the most well-established methods to study SOCE is using the Ca2+-sensing dye, fura-2. Here we describe a detailed protocol on how to use fura-2 to study Ca2+ signaling from SOCE in human embryonic kidney (HEK) cells.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.