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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 (IC 50 : 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., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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.

Orai1 is the pore-forming subunit of the store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1β, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca 2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca 2+ concentration, as demonstrated by cell loading with the fast intracellular Ca 2+ chelator dimethyl BAPTA in the absence of extracellular Ca 2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1β to the plasma membrane when expressed individually; by contrast, when Orai1β is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1β in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca 2+ store depletion., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Ethnopharmacological Relevance: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive., Aims of the Study: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively., Materials and Methods: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR., Results: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 μM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 μM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR., Conclusion: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Regardless of its aetiology, sustained intracellular Ca 2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca 2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca 2+ influx channel, is known to contribute to sustained Ca 2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca 2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca 2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca 2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca 2+ channel that mediates extracellular Ca 2+ influx upon endoplasmic reticulum Ca 2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca 2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca 2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Calcium signaling plays a vital role in the regulation of various cellular processes, including activation, proliferation, and differentiation of T-lymphocytes, which is mediated by ORAI1 and potassium (K + ) channels. These channels have also been identified as highly attractive therapeutic targets for immune-related diseases. Licochalcone A is a licorice-derived chalconoid known for its multifaceted beneficial effects in pharmacological treatments, including its anti-inflammatory, anti-asthmatic, antioxidant, antimicrobial, and antitumorigenic properties. However, its anti-inflammatory effects involving ion channels in lymphocytes remain unclear. Thus, the present study aimed to investigate whether licochalcone A inhibits ORAI1 and K + channels in T-lymphocytes. Our results indicated that licochalcone A suppressed all three channels (ORAI1, Kv1.3, and KCa3.1) in a concentration-dependent matter, with IC 50 values of 2.97 ± 1.217 µM, 0.83 ± 1.222 µM, and 11.21 ± 1.07 µM, respectively. Of note, licochalcone A exerted its suppressive effects on the IL-2 secretion and proliferation in CD3 and CD28 antibody-induced T-cells. These results indicate that the use of licochalcone A may provide an effective treatment strategy for inflammation-related immune diseases.

Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca 2+ -channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca 2+  entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca 2+ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 μM) or SGK1 inhibitor GSK-650394 (1 μM). Transcript levels were measured using q-RT-PCR, cytosolic Ca 2+ -concentration ([Ca 2+ ] i ) by Fura-2-fluorescence, and store-operated Ca 2+ entry from increase of [Ca 2+ ] i following re-addition of extracellular Ca 2+ after store depletion with thapsigargin (1 μM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca 2+ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca 2+ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca 2+  entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca 2+ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca 2+  entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca 2+ -deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca 2+ -deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: • In HAoSMCs, vasopressin (VP) upregulates Ca 2+ channel ORAI1 and its activator STIM1. • VP upregulates store-operated Ca 2+  entry (SOCE) and osteogenic signalling (OS). • VP-induced SOCE, OS and Ca 2+ -deposition are disrupted by ORAI1 inhibitor MRS1845. • VP-induced SOCE, OS and Ca 2+ -deposition are disrupted by SGK1 blocker GSK-650394.

This study was undertaken to test two therapies for acute kidney injury (AKI) prevention, IGF-1, which is renal protective, and BTP-2, which is a calcium entry (SOCE) inhibitor. We utilized lipopolysaccharide (LPS) IP, as a systemic model of AKI and studied in five groups of animals. Three experiments showed that at 7 days: (1) LPS significantly reduced serum IGF-1 and intramuscular IGF-I in vivo gene therapy rescued this deficiency. (2) Next, at the 7-day time point, our combination therapy，compared to the untreated group,caused a significant increase in survival, which was noteworthy because all of the untreated animals died in 72 hrs. (3) The four pathways associated with inflammation, including (A) increase in cytosolic calcium, (B) elaboration of proinflammatory cytokines, (C) impairment of vascular integrity, and (D) cell injury, were adversely affected in renal tissue by LPS, using a sublethal dose of LPS. The expression of several genes was measured in each of the above pathways. The combined therapy of IGF-1 and BTP-2 caused a favorable gene expression response in all four pathways. Our current study was an AKI study, but these pathways are also involved in other types of severe inflammation, including sepsis, acute respiratory distress syndrome, and probably severe coronavirus infection.

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca 2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H 2 O 2 , 10-100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H 2 O 2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca 2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca 2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear., Methods: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1 R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery., Results: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn 2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca 2+ signaling alterations (increased SOCE, decreased [Ca 2+ ] i transients amplitude and decay rate, lower SR Ca 2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult., Conclusions: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Calcium (Ca 2+ ) and its relevant transmembrane and intracellular calcium channels were previously thought to be chiefly associated with the regulation of cardiovascular and neuronal systems. Nowadays, an increasing evidence shows those calcium channels are also responsible for tumorigenesis and progression. However, the general underlying mechanisms and the involving signaling transduction pathways still remain unclear. Therefore, in this mini-review, we are mainly focusing on the linkage between calcium channels and major characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, evasion of immune surveillance, and the alterations of tumor microenvironment. We will shed light on the possible therapeutic approaches to counteract tumors regarding the intervention of calcium channel., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Purpose: Mibefradil, a T-type Ca 2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca 2+ channels., Experimental Approach: Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca 2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry., Key Results: Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC 50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 μM respectively. Outside-out patch demonstrated that perfusion of 10-μM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 μM) inhibited Ca 2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca 2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression., Conclusions and Implications: Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity., (© 2019 The British Pharmacological Society.)

Key Points: This work confirms previous reports that CM4620, a small molecule inhibitor of Ca 2+ entry via store operated Ca 2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells., Abstract: Key features of acute pancreatitis include excess cellular Ca 2+ entry driven by Ca 2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca 2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca 2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca 2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Background: Dysregulation in calcium (Ca 2+ ) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca 2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca 2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca 2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study., Methods: An extensive analysis of the Ca 2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca 2+ entry, basal Ca 2+ levels, and store operated Ca 2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients., Results: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca 2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP 3 R (SOCE) Ca 2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1 PM ); and (vi) blocked when using a mAb targeting STIM1 PM . Next, we further established an association between an elevated expression of STIM1 PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1 PM CLL subgroup., Conclusions: These data establish the critical role of a newly discovered BCR independent Ca 2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca 2+ entry by enhancing store-operated Ca 2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca 2+ -induced Lats1/2 activation. Ca 2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca 2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca 2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.

Key Points: Gain-of-function mutations in the highly selective Ca 2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca 2+ influx and increasing the currents evoked by Ca 2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca 2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM., Abstract: Skeletal muscle differentiation relies on store-operated Ca 2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca 2+ stores to the activation of membrane Ca 2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca 2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca 2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca 2+ ions and conferred resistance to acidic inhibition. Ca 2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1 -/- /Stim2 -/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Objective: To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM., Results: The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca 2+  release-activated Ca 2+ /calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs., Conclusion: ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs., (© 2018, American College of Rheumatology.)

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit + progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca 2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit + progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca 2+ ( Ca i 2 + ) by triggering a transient Ca 2+ release from ER IP3Rs followed by sustained Ca 2+ influx through store-operated Ca 2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca 2+ release and Ca 2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Ca i 2 + via ER-IP3R3 Ca 2+ release followed by Ca 2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit + progenitor cells., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The proteins Orai1 and STIM1 control store-operated Ca 2+ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca 2+ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Store operated calcium channels (SOCCs) have been suggested to play a critical role in many diabetic complications. Diabetic cystopathy (DCP) is common in patients with diabetes, but the role of SOCCs in DCP is still unclear. The aim of the present study was to investigate the role of SOCCs in DCP with streptozocin (STZ)‑induced diabetic rats. Specifically, the authors investigated whether SOCCs were altered in streptozocin (STZ)‑induced diabetic rats and, if so, how this may contribute to the contraction of bladder detrusor strips and the intracellular Ca2+ concentration of bladder smooth muscle cells in diabetic rats. Cyclopiazonic acid (CPA, 10 µM) and SKF‑96365 (10 µM) were used to activate and inhibit SOCCs respectively, to research the effects of SOCCs on the contraction of the bladder detrusor strips in normal and STZ‑induced diabetic rats at the 4th, 8th and 12th week after the diabetic rat model was established. The changes of intracellular Ca2+ were also evaluated under confocal microscopy with pretreated Fluo‑4AM. In addition, the expressions of Orai1 and STIM1 were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting at different time points. According to the results, the contractive frequency of diabetic bladder muscle strips was higher than that of controls in the 4th and 8th week. The increased fluorescence intensity was detected after using CPA and SKF‑96365 in diabetic groups. The expressions of Orai1 and STIM1 changed in a time‑dependent manner.

Ca 2+ entry mediated by the calcium channel, Orai1, provides critical Ca 2+ signals that regulate cell function. The ER-Ca 2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca 2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca 2+ ]. The increase in Ca 2+ entry controls Ca 2+ -dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca 2+ signaling and the physiological response of cells., (Published by Elsevier Inc.)

The role of Ca 2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca 2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca 2+ , while both cell lines showed marked reduced levels of Ca 2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca 2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca 2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca 2+ ], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca 2+ homeostasis and that such changes favor viral replication., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca 2+ entry (SOCE) and diminished Ca 2+ -dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca 2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca 2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.

Background: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin., Objective and Methods: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE., Results: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1., Conclusions: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca 2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca 2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N , N , N' , N' -tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn 2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn 2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca 2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca 2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1., (© FASEB.)

Background/aims: Multiple myeloma (MM) is a plasma cell neoplasm which constitutes about 10% of all hematologic malignancies. Despite the development and application of novel agents, MM still undergoes an aggressive and incurable course in the vast majority of patients. Ca2+ is one of the critical regulators of cell migration. Ca2+ influx is essential for the migration of various types of cells including tumor cells. However, the role of store-operated calcium entry (SOC) channels, the only Ca2+ channels of non-excitable cells, has not yet been reported in MM cell survival., Methods: We evaluated the expression of Stim1 and Orai1 (two key regulators of SOC) in MM tissues and cell lines by immunohistochemical assay, quantitative real-time PCR assay and western blot. MM cell lines were pretreated with pharmacological blockers and siRNAs, and then MM cell proliferation, cell cycle arrest, and apoptosis were examined by FACS (flow cytometry) assay, and Annexin V-FITC/PI staining. The correlation between the expression of Stim1 (or Orai1) level and outcome in MM were assessed by using Progress Free Survival (PFS)., Results: Stim1 and Orai1 were both abundantly expressed in MM tissue and MM cell lines. Inhibition of SOCE reduced MM cell viability, and induced cell cycle arrest and apoptosis. Stim1 or Orai1 silencing also reduced cell viability, caused cell apoptosis and cell cycle arrest in MM cell lines. Over-expression of Stim1/Orai1 in MM patients was closely associated with the clinical outcome of MM., Conclusion: The Stim1/Orai1-mediated signaling participates in the pathogenesis of MM, which represents an attractive target for future therapeutic intervention., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca 2+ -dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca 2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.

Context: Spirodela polyrhiza (L.) Schleid. (Lemnaceae), Spirodelae Herba (SH), has been known to relieve inflammation, urticaria and skin symptoms including pruritus, eczema and rash., Objective: The effects of SH extract on two calcium ion channels, Orai1 and TRPV3, and their potential as novel therapeutics for atopic dermatitis (AD) were investigated. The regulatory role of Orai1 on mast cell degranulation was evaluated., Materials and Methods: The dried leaves of SH were extracted by 70% methanol. Effects of SH extract (100 μg/mL) in an HEK293T cell line overexpressing human Orai1 or TRPV3 were assessed. Ion channel modulation in transfected HEK293T cells was measured using a conventional whole-cell patch-clamp technique. IgE-antigen complex-stimulated mast cell degranulation was measured by β-hexosaminidase assay with morphological observation after treatment with 20, 50 and 100 μg/mL SH extract., Results: SH extract (100 μg/mL) significantly inhibited Orai1 activity (63.8 ± 0.97%) in Orai1-STIM1 co-overexpressed HEK293T cells. SH extract significantly increased TRPV3 activity (81.29 ± 0.05% at -100 mV) compared with the positive control 2-APB (100 μM), which induced full activation. SH extract inhibited degranulation in IgE-antigen complex-stimulated RBL-2H3 mast cells by decreasing β-hexosaminidase activity (3.14 ± 0.03, 2.56 ± 0.12 and 2.29 ± 0.08 mU/mg, respectively)., Conclusion: Our results suggested that SH extract could treat abnormal skin barrier pathologies in AD through modulation of the activities of the calcium ion channels Orai1 and TRPV3 and inhibition of mast cell degranulation. This is the first report of an herbal effect on the modulation of ion channels associated with skin barrier disruption in AD pathogenesis.

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca 2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca 2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca 2+ levels, respectively. Histamine release and Ca 2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca 2+ increase. FcεRI-induced Ca 2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca 2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca 2+ influx enhanced the process. Furthermore, cytosolic Ca 2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca 2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca 2+ signaling in regulating histamine degranulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca 2+ entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca 2+ channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La 3+ ; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca 2+ channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca 2+ channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway., (Copyright © 2017 the American Physiological Society.)

Collagen IV (Col IV) is a major component of expanded glomerular extracellular matrix in diabetic nephropathy and Smad1 is a key molecule regulating Col IV expression in mesangial cells (MCs). The present study was conducted to determine if Smad1 pathway and Col IV protein abundance were regulated by store-operated Ca 2+ entry (SOCE). In cultured human MCs, pharmacological inhibition of SOCE significantly increased the total amount of Smad1 protein. Activation of SOCE blunted high-glucose-increased Smad1 protein content. Treatment of human MCs with ANG II at 1 µM for 15 min, high glucose for 3 days, or TGF-β1 at 5 ng/ml for 30 min increased the level of phosphorylated Smad1. However, the phosphorylation of Smad1 by those stimuli was significantly attenuated by activation of SOCE. Knocking down Smad1 reduced, but expressing Smad1 increased, the amount of Col IV protein. Furthermore, activation of SOCE significantly attenuated high-glucose-induced Col IV protein production, and blockade of SOCE substantially increased the abundance of Col IV. To further verify those in vitro findings, we downregulated SOCE specifically in MCs in mice using small-interfering RNA (siRNA) against Orai1 (the channel protein mediating SOCE) delivered by the targeted nanoparticle delivery system. Immunohistochemical examinations showed that expression of both Smad1 and Col IV proteins was significantly greater in the glomeruli with positively transfected Orai1 siRNA compared with the glomeruli from the mice without Orai1 siRNA treatment. Taken together, our results indicate that SOCE negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in MCs., (Copyright © 2017 the American Physiological Society.)

The concentration of free cytosolic Ca 2+ and the voltage across the plasma membrane are major determinants of cell function. Ca 2+ -permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca 2+ -permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca 2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca 2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Orai1 Ca 2+ permeable ion channel is an important component of store operated Ca 2+ entry (SOCE) in cells. It's over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure-activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background/aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells., Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements., Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation., Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: Ultraviolet radiation exposure is the most important cause of extrinsic skin aging (photoaging), which causes skin wrinkling and hyperpigmentation. Although many factors are involved in the photoaging process, calcium release-activated calcium channel protein 1 (ORAI1) has been reported to be involved in UV-induced melanogenesis., Objective: The aim of the present study was to find inhibitory effects of the extract of Foeniculum vulgare (fennel) fruits on ORAI1 ion channels and UV-induced melanogenesis in melanoma cells and to identify its active constituents., Methods: Active compounds were isolated and quantitatively analyzed. An electrophysiological assay was performed by using the whole-cell patch-clamp technique. Intracellular free calcium concentration was measured by Fura-2. Tyrosinase activity was evaluated by levodopa colorimetry. Effects of the most active compound on cell viability of murine B16F10 melanoma cells and inhibition of melanin content after UVB irradiation were determined., Results: F. vulgare fruits extract and its hexane fraction strongly blocked ORAI1 currents and tyrosinase activity and significantly inhibited UV-induced melanogenesis. Of the 13 compounds isolated from the hexane fraction, trans-anethole (TA) exhibited inhibitory effects on ORAI1 (IC 50 =8.954±1.36μM) and increased cytoplasmic Ca 2+ concentrations in response. TA inhibited UV-induced melanogenesis without affecting tyrosinase activity., Conclusion: Our findings suggest that the fruits extract of F. vulgare and its active constituent, TA, provide a possible novel approach for treating and preventing UV-induced melanogenesis., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Calcium release-activated calcium modulator 1(ORAI1) is an integral component of the calcium release-activated calcium channel (CRAC) channel complex and plays a central role in regulating Ca 2 +  concentrations in T-lymphocytes. It is critical for many physiological processes, including cell-proliferation, cytokine production and activation of the immune system. Loss of ORAI1 function is linked with rheumatoid arthritis (RA) and hence pharmacological blockers of ORAI1 could be potential therapeutic agents for the treatment of RA. In this study, we have used a high-throughput screening approach to inhibit the binding of Ca 2+  toward ORAI1 and the interactions are verified through induced fit docking. The results hint that these compounds act by possibly binding with, and thereby blocking Ca 2+ -binding with ORAI1 (E106). The molecular dynamics (MD) simulations shows strong support toward the hit compounds by showing the ligand potency throughout the simulation timescale of 30 ns. We have thus identified a novel class of highly stable, potential lead compounds that directly bind with the selectivity filter region E106 and block Ca 2+ binding on ORAI1. This resulting alteration in the pore geometry of ORAI1 due to the strong blocking mechanism of lead compounds will greatly diminish its function and the downstream activities that result from the same including decreased production of cytokines in autoimmune disorders. This study may lay the foundation for finding novel lead compounds for clinical trials that could positively modulate the course of autoimmune disorders with ORAI1 as its specific target.

Ultraviolet (UV) irradiation is a major environmental factor affecting photoageing, which is characterized by skin wrinkle formation and hyperpigmentation. Although many factors are involved in the photoageing process, UV irradiation is thought to play a major role in melanogenesis. Tyrosinase is the key enzyme in melanin synthesis; therefore, many whitening agents target tyrosinase through various mechanisms, such as direct interference of tyrosinase catalytic activity or inhibition of tyrosinase mRNA expression. Furthermore, the highly selective calcium channel ORAI1 has been shown to be associated with UV-induced melanogenesis. Thus, ORAI1 antagonists may have applications in the prevention of melanogenesis. Here, we aimed to identify the antimelanogenesis agents from methanolic extract of guava leaves (Psidium guajava) that can inhibit tyrosinase and ORAI1 channel. The n-butanol (47.47%±7.503% inhibition at 10 μg/mL) and hexane (57.88%±7.09% inhibition at 10 μg/mL) fractions were found to inhibit ORAI1 channel activity. In addition, both fractions showed effective tyrosinase inhibitory activity (68.3%±0.50% and 56.9%±1.53% inhibition, respectively). We also confirmed that the hexane fraction decreased the melanin content induced by UVB irradiation and the ET-1-induced melanogenesis in murine B16F10 melanoma cells. These results suggest that the leaves of P. guajava can be used to protect against direct and indirect UV-induced melanogenesis., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Evidence supports an important role of Ca 2+ release-activated Ca 2+ channel protein 1 (Orai1)-mediated Ca 2+ entry in the development of renal fibrosis, a common pathologic feature of CKDs that lead to ESRD, but the molecular mechanisms remain unclear. We determined the role of Orai1 calcium channel in renal fibrosis induced by high-fat diet and by unilateral ureteral obstruction. Mouse kidneys with fibrosis had higher levels of Orai1 protein expression than did kidneys without fibrosis. In vivo knockdown of Orai1 with adenovirus harboring Orai1-short hairpin RNA or inhibition of Orai1 with SKF96365 dramatically prevented renal fibrosis and significantly decreased protein expression of fibronectin, α‑smooth muscle actin, and TGF‑β1 in the kidney cortex of ApoE -/- mice on a high-fat diet and in the obstructed kidneys of mice with unilateral ureteral obstruction. Compared with kidney biopsy specimens of patients with glomerular minimal change disease, those of patients with fibrotic nephropathy had higher expression levels of Orai1. In cultured human proximal tubule epithelial cells (HK2), knockdown of Orai1 Ca 2+ channel with adenovirus-Orai1-short hairpin RNA markedly inhibited TGF-β1-induced intracellular Ca 2+ influx and phosphorylation of smad2/3. Knockdown or blockade of the Orai1 Ca 2+ channel in HK2 cells also prevented epithelial-to-mesenchymal transition induced by TGF‑β1. In conclusion, blockade of the Orai1 Ca 2+ channel prevented progression of renal fibrosis in mice, likely by suppressing smad2/3 phosphorylation and TGF-β1-induced epithelial-to-mesenchymal transition. These results render the Orai1 Ca 2+ channel a potential therapeutic target against renal fibrosis., (Copyright © 2016 by the American Society of Nephrology.)

Store-operated Ca(2+) entry mediated by STIM1-gated Orai1 channels is essential to activate immune cells and its inhibition or gain-of-function can lead to immune dysfunction and other pathologies. Reactive oxygen species interacting with cysteine residues can alter protein function. Pretreatment of the Ca(2+) selective Orai1 with the oxidant H2O2 reduces ICRAC with C195, distant to the pore, being its major redox sensor. However, the mechanism of inhibition remained elusive. Here we combine experimental and theoretical approaches and show that oxidation of Orai1 leads to reduced subunit interaction, slows diffusion and that either oxidized C195 or its oxidomimetic mutation C195D located at the exit of transmembrane helix 3 virtually eliminates channel activation by intramolecular interaction with S239 of transmembrane helix 4, thereby locking the channel in a closed conformation. Our results demonstrate a novel mechanistic model for ROS-mediated inhibition of Orai1 and identify a candidate residue for pharmaceutical intervention.

Background: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes., Methods: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay., Results: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability., Conclusions: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Aberrant Ca(2+) release-activated Ca(2+) (CRAC) channel activity has been implicated in a number of human disorders, including immunodeficiency, autoimmunity, occlusive vascular diseases and cancer, thus placing CRAC channels among the important targets for the treatment of these disorders. We briefly summarize herein the molecular basis and activation mechanism of CRAC channel and focus on discussing several pharmacological inhibitors of CRAC channels with respect to their biological activity, mechanisms of action and selectivity over other types of Ca(2+) channel in different types of cells.

An increase in intracellular Ca2+ concentration plays a key role in the establishment of many cancer hallmarks, including aberrant proliferation, migration, invasion, resistance to apoptosis and angiogenesis. The dysregulation of Ca2+ entry is one of the most subtle mechanisms by which cancer cells overwhelm their normal counterparts and gain the adaptive advantages that result in tumour growth, vascularisation and dissemination throughout the organism. Both constitutive and agonist-induced Ca2+ influx may be mediated by store-dependent as well as store-independent Ca2+ entry routes. A growing body of evidences have shown that different isoforms of Stromal Interaction Molecules (Stim1) and Orai proteins, i.e. Stim1, Stim2, Orai1 and Orai3, underlie both pathways in cancer cells. The alteration in either the expression or the activity of Stim and Orai proteins has been linked to the onset and maintenance of tumour phenotype in many solid malignancies, including prostate, breast, kidney, esophageal, skin, brain, colorectal, lung and liver cancers. Herein, we survey the existing data in support of Stim and Orai involvement in tumourigenesis and provide the rationale to target them in cancer patients. Besides, we summarize the most recent advances in the identification of novel pharmacological tools that could be successfully used in clinical therapy.