Capacitative Calcium Entry Research Articles

Ciguatera poisoning: the role of high-voltage-activated and store-operated calcium channels in ciguatoxin-induced sensory effects

Introduction: Ciguatera fish poisoning (CFP), the most common seafood poisoning worldwide, is caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Pruritus is one of the most distressing symptoms, associated with other cutaneous sensory disorders, including paresthesia and cold dysesthesia. No specific treatment exists. CTXs are known to primarily activate voltage-gated sodium channels, but the downstream molecular events that lead to sensory disturbances remain poorly defined. Peptidergic sensory neurons were recently identified as major players in CFP sensory disturbances. Methods: In this study, we examined the role of molecular actors in 2 effects induced by Pacific CTX-2 (P-CTX-2): the increase in cytosolic calcium levels in rat primary sensory neurons; and the release of the neuropeptide substance P (SP) in sensory neurons co-cultured with keratinocytes. Results: Our results (i) rule out the involvement of the Na+/Ca2+ exchanger (NCX) and the transient receptor potential channels transient receptor potential ankyrin 1 and and transient receptor potential vanilloid 1; (ii) show that N-type voltage-gated calcium (Cav) channels contribute to the initiation of the calcium signal elicited by P-CTX-2 in rat sensory neurons, while N-type and L-type Cav channels play equal parts in the SP release in the co-culture; and (iii) identify store-operated calcium entry supported by Orai calcium release-activated calcium modulator 1 (ORAI1) as a critical effector of the late phase of the calcium signal and the subsequent SP release elicited by P-CTX-2. Discussion: Our in vitro findings indicate that Cav and ORAI1 channels may be promising pharmacological targets for specifically relieving the sensory effects of CTXs.

ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

Gating and regulation of the calcium release-activated calcium channel: Recent progress from experiments and molecular modeling.

Calcium release-activated calcium (CRAC) channels are highly calcium ion (Ca2+)-selective channels in the plasma membrane. The transient drop of endoplasmic reticulum Ca2+ level activates its calcium sensor stromal interaction molecule (STIM) and then triggers the gating of the CRAC channel pore unit Orai. This process involves a variety of activities of the immune system. Therefore, understanding how the activation and regulation of the CRAC channel can be accomplished is essential. Here we briefly summarize the recent progress on Orai gating and its regulation by 2-aminoethoxydiphenylborate (2-APB) obtained from structural biology studies, biochemical and electrophysiological measurements, as well as molecular modeling. Indeed, integration between experiments and computations has further deepened our understanding of the channel gating and regulation.

Effects of store-operated and receptor-operated calcium channels on synchronization of calcium oscillations in astrocytes

Intercellular calcium signaling allows cells to communicate with each other and to interact with adjacent cells. Gap junction is the most common and important way for cellular communication. Recently, mathematical models have been widely used to gain a precise and quantitative understanding of the dynamics of intracellular calcium ions (Ca2＋). In this paper, we establish a mathematical model considering the gap junction permeable to Ca2＋ and to IP3 for describing the calcium oscillations in coupled astrocytes. Store-operated calcium entry (SOCE) is viewed as the main process which controls the non-excitable cells, hence, we focus on the effect of store-operated calcium channel (SOCC) and receptor-operated calcium channel (ROCC) on the intercellular synchronization, respectively. By employing bifurcation analysis on this model, the dynamic behaviors of the coupled system with different physiological state cells is obtained with changes in the maximum capacity of the SOCC and the ROCC. The synchronization boundaries for different conditions are gained in the two parameters space of the channel parameters and the coupling strength. The results suggest that the variation of the maximum flow for different calcium channels determines the stable oscillations of the coupled system, as well as for the frequency and amplitude of oscillations. The SOCC has an expected effect on the change of the oscillatory interval while the ROCC demonstrated the influence on the amplitude modulation. Furthermore, the coupling strength and channel parameters could induce 1:1 locking of intercellular Ca2＋ oscillations and the synchronization region like Arnol’d tongue is found.

The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca2+)c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O2•–) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca2+)c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Comprehensive Construction of a Circular RNA-Associated Competing Endogenous RNA Network Identified Novel Circular RNAs in Hypertrophic Cardiomyopathy by Integrated Analysis.

Hypertrophic cardiomyopathy (HCM), the most common heritable cardiomyopathy, is associated with a high risk of sudden cardiac death. The complexity and behavior of the circular RNA (circRNA)-associated competing endogenous RNA (ceRNA) network in HCM have not been thoroughly elucidated. Plasma circRNA and messenger RNA (mRNA) expression profiles were acquired by using a microarray. Weighted correlation network analysis (WGCNA) and linear models for microarray data (Limma) were used to analyze microarray data. Gene modules, consisting of genes with high correlations, were detected and represented by a designated color. The ceRNA network, including circRNA, microRNA (miRNA), and mRNA, was constructed based on the “ceRNA hypothesis” using an integrated systems biology method. By WGCNA, two modules, namely magenta and red modules, were identified as being positively correlated with HCM. In the combined analysis of WGCNA and Limma, 36 hub circRNAs in the magenta module and 83 hub circRNAs in the red module were significantly upregulated compared with the controls. By coexpression analysis, 270 circRNA–mRNA pairs were identified with a coefficient ≥0.9 and p < 0.05. With Starbase and miRWalk tools, circRNA–miRNA pairs and miRNA–mRNA pairs were predicted. Once these pairs were combined, the ceRNA network with 6 circRNAs, 29 miRNAs, and 6 mRNAs was constructed. Functional analysis demonstrated that these circRNAs in the ceRNA network were associated with calcium-release channel activity and muscle filament sliding. Our study provided a global perspective and systematic analysis of the circRNA-associated ceRNA network in HCM. The identified circRNAs hsa_circ_0043762, hsa_circ_0036248, and hsa_circ_0071269 may be key regulators involved in HCM pathogenesis.

Mechanism of thromboxane receptor-induced vasoconstriction in human saphenous vein

Saphenous vein (SV) is one of the most widely used graft material in patients undergoing coronary artery bypass graft surgery (CABG). Thromboxane A2 (TXA2) is implicated in graft failure by inducing vasoconstriction and platelet aggregation. The aim of this study is to investigate the mechanism involved in TXA2-induced vasoconstriction in human SV. The role of different inhibitors and blockers on U46619 (TXA2-mimetic)-induced vasoconstriction is investigated by using an isolated organ bath system. Relaxation responses to several mediators are evaluated in SV pre-contracted with U46619 and compared with those pre-contracted with phenylephrine. Our results demonstrate that U46619-induced contraction is completely blocked by myosin light chain kinase inhibitor ML-9 or TP receptor antagonist BAY u3405. Furthermore, U46619-induced contraction is partially inhibited by phospholipase C inhibitor U73122, protein kinase C inhibitor calphostin C, Rho-kinase inhibitor Y-27632, L-type calcium channel blocker nifedipine, store-operated channel inhibitor SKF96365 or removal of extracellular calcium. Relaxation responses to NO donor (sodium nitroprusside), guanylate cyclase (GC) stimulator (riociguat), phosphodiesterase (PDE) inhibitors (sildenafil, IBMX), adenylate cyclase (AC) activator (forskolin) and acetylcholine (ACh) are markedly reduced when U46619 is used as a pre-contraction agent. Our results demonstrate that influx of extracellular Ca2+ (through L-type calcium channels and store-operated calcium channels) and intracellular Ca2+ release together with Ca2+ sensitization (through Rho-kinase activation) are necessary components for TXA2-induced vasoconstriction in SV. Moreover, more pronounced decrease in vasorelaxation induced by several mediators (SNP, riociguat, sildenafil, IBMX, forskolin, and ACh) in the presence of U46619 when compared with phenylephrine suggests that there is a crosstalk between the TP receptor signaling pathway and PDE, AC, GC enzymes. We believe that the investigation of mechanism of the TXA2-induced vasoconstriction in SV will provide additional information for the prevention of SV graft failure.

Anti-ORAI1 antibody DS-2741a, a specific CRAC channel blocker, shows ideal therapeutic profiles for allergic disease via suppression of aberrant T-cell and mast cell activation.

ORAI1 constitutes the pore‐forming subunit of the calcium release‐activated calcium (CRAC) channel, which is responsible for store‐operated calcium entry into lymphocytes. It is known that ORAI1 is essential for the activation of T cells and mast cells and is considered to be a potent therapeutic target for autoimmune and allergic diseases. Here, we obtained a new humanized antibody, DS‐2741a, that inhibits ORAI1 function. DS‐2741a bound to human‐ORAI1 with high affinity and without cross‐reactivity to rodent Orai1. DS‐2741a demonstrated suppression of CRAC‐mediated human and mouse T‐cell activation and mast cell degranulation in human ORAI1 knock‐in mice. Furthermore, DS‐2741a ameliorated house dust mite antigen‐induced dermatitis in the human ORAI1 knock‐in mouse. Taken together, DS‐2741a inhibited T‐cell and mast cell functions, thus improving skin inflammation in animal models of atopic dermatitis and reinforcing the need for investigation of DS‐2741a for the treatment of allergic diseases in a clinical setting.

Functional coupling between BKCa and SOC channels

High-conductance, voltage- and calcium-activated potassium channels (BKCa) and store-operated calcium channels (SOCs) are belong to K+ channels superfamily and calcium channels superfamily respectively. Since BKCa potassium channels can be activated by calcium ions, we set out to examine whether SOCs are coupled with BKCa and to probe the relationship of BKCa and SOCs. First, we proved that BKCa immunoprecipitated with Orai1, and confocal microscopy showed that BKCa co-localized with Orai1. Next, we mapped that the exact binding sites locate in the 350aa-371aa fragment of the first regulatory domain associated with K+ conduction (RCK1) and the 720aa-814aa fragment in the second regulatory domain associated with K+ conduction (RCK2) via GST-pull-down assays. Then, we showed, by calcium imaging that BKCa overexpression enhanced endogenous and exogenous store-operated calcium entry (SOCE) and this enhancement could be blocked by Orai1 knockdown. Finally, we proved that SOCE could enhance the activity of BKCa by patch-clamp. From these results we conclude that BKCa can form a positive feedback loop with SOCs, as the Ca2+ influx from SOCs can active BKCa, which can hyperpolarize the plasma membrane. In turn, the hyperpolarized membrane will form a higher electric potential difference and give more force, allowing Ca2+ influx via SOCs.

Targeting Calcium Release-activated Calcium Channel Is Not Sufficient to Prevent Rejection in Nonhuman Primate Kidney Transplantation.

Calcineurin inhibitors successfully control rejection of transplanted organs but also cause nephrotoxicity. This study, using a rhesus monkey renal transplantation model, sought to determine the applicability of a new immunomodulatory drug inhibiting the store-operated calcium release-activated calcium channel of lymphocytes to control transplant rejection without nephrotoxicity. Animals underwent kidney transplantation and were treated with tacrolimus alone (n = 3), a CRACM1 inhibitor (PRCL-02) (n = 6) alone, or with initial tacrolimus monotherapy followed by gradual conversion at 3 weeks to PRCL-02 alone (n = 3). PRCL-02 was administered via a surgically inserted gastrostomy tube BID. Dose-related drug exposure in monkeys was established and renal transplants were then performed using PRCL-02 monotherapy. Oral dosing of PRCL-02 was well tolerated and resulted in suppressed T-cell proliferation in in vitro MLR comparable to animals in the tacrolimus control arm. Animals receiving tacrolimus monotherapy were e on day 100 without rejection. PRCL-02 monotherapy only marginally prolonged graft survival (MST = 13.16 d; group 2) compared with untreated controls. Animals treated initially with tacrolimus and converted to PRCL-02 monotherapy had a mean graft survival of 35.3 days which was prolonged compared with PRCL-02 monotherapy but not compared with the tacrolimus-treated group. Pharmacokinetic studies showed inconsistent drug exposures despite attempts to adjust dose and exposure which may have contributed to the rejections. We conclude that, in this nonhuman primate model of kidney transplantation, PRCL-02 demonstrated evidence of in vivo immunosuppressive activity but was inferior to tacrolimus treatment with respect to suppressing immune transplant rejection.

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.

Calcium Release-Activated Calcium (CRAC) Channel Inhibition Suppresses Pancreatic Ductal Adenocarcinoma Cell Proliferation and Patient-Derived Tumor Growth.

Pancreatic ductal adenocarcinoma (PDAC) remains an unmet clinical problem in urgent need of newer molecularly driven treatment modalities. Calcium signals, particularly those associated with calcium release-activated calcium (CRAC) channels, are known to influence the development, growth, and metastasis of many cancers. This is the first study investigating the impact of CRAC channel inhibition on PDAC cell lines and patient-derived tumor models. PDAC cell lines were exposed to a novel CRAC channel inhibitor, RP4010, in the presence or absence of standard of care drugs such as gemcitabine and nab-paclitaxel. The in vivo efficacy of RP4010 was evaluated in a hyaluronan-positive PDAC patient-derived xenograft (PDx) in the presence or absence of chemotherapeutic agents. Treatment of PDAC cell lines with single-agent RP4010 decreased cell growth, while the combination with gemcitabine/nab-paclitaxel exhibited synergy at certain dose combinations. Molecular analysis showed that RP4010 modulated the levels of markers associated with CRAC channel signaling pathways. Further, the combination treatment was observed to accentuate the effect of RP4010 on molecular markers of CRAC signaling. Anti-tumor activity of RP4010 was enhanced in the presence of gemcitabine/nab-paclitaxel in a PDAC PDx model. Our study indicates that targeting CRAC channel could be a viable therapeutic option in PDAC that warrants further clinical evaluation.

Inhibition of store-operated calcium channels by N-arachidonoyl glycine (NAGly): no evidence for the involvement of lipid-sensing G protein coupled receptors

N-arachidonoyl glycine (NAGly) is an endogenous lipid deriving from the endocannabinoid anandamide (AEA). Identified as a ligand of several G-protein coupled receptors (GPCRs), it can however exert biological responses independently of GPCRs. NAGly was recently shown to depress store-operated Ca2+ entry (SOCE) but its mechanism of action remains elusive. The major aim of this study was to gain a better knowledge on the NAGly-dependent impairment of SOCE in neurons of the central nervous system (CNS) from mice. First, we examined the expression of genes encoding for putative lipid sensing GPCRs using transcriptomic data publicly available. This analysis showed that the most abundant GPCRs transcripts present in the cerebral cortices of embryonic brains were coding for lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) receptors. Next, the presence of functional receptors was assessed with live-cell calcium imaging experiments. In primary cortical cells S1P and LPA mobilize Ca2+ from internal stores via a mechanism sensitive to the S1P and LPA receptor antagonists Ex26, H2L5186303, or Ki16425. However, none of these compounds prevented or attenuated the NAGly-dependent impairment of SOCE. We found no evidence for the requirement of lipid sensing GPCRs in this inhibitory process, indicating that NAGly is an endogenous modulator interfering with the core machinery of SOCE. Moreover, these data also raise the intriguing possibility that the depression of SOCE could play a role in the central effects of NAGly.

The Blockade of Store-Operated Calcium Channels Improves Decompression Sickness in Rats.

BackgroundPrevious investigations reveal that BTP2, a store-operated calcium channel blocker, has protective and anti-inflammatory properties in multiple inflammatory diseases. This study investigates whether BTP2 can protect against decompression sickness (DCS) in a rat model.MethodsBTP2 (2 mg/kg) was administered to male Sprague–Dawley rats 30 min before subjecting them to hyperbaric pressure. Control rats were not treated. After decompression, signs of DCS were examined, and samples of bronchoalveolar lavage fluid and lung tissue were obtained for evaluation.ResultsThe incidence and mortality of DCS were decreased significantly in rats treated with BTP2 compared to those treated with dimethyl sulfoxide. BTP2 significantly attenuated DCS-induced lung edema, histological evidence of lung inflammation, necroptosis, and apoptosis, while it decreased levels of tumor necrosis factor alpha, interleukin-6, and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluid. In addition, BTP2 reduced the expression of nuclear factor of activated T cells and early growth response protein 3 in lung tissue. BTP2 also significantly increased the levels of inhibitor kappa B alpha and suppressed the levels of nuclear factor kappa B in lung tissue.ConclusionThe results suggest that BTP2 may has potential as a prophylactic therapy to attenuate DCS-induced injury.

Insights into Activation Mechanisms of Store-Operated TRPC1 Channels in Vascular Smooth Muscle.

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

Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion

Ischemia and Reperfusion (I/R) injuries are associated with coronary artery hypercontracture. They are mainly originated by an exacerbated response to agonists released by endothelium such as Endothelin (ET-1), involving the alteration in intracellular calcium handling. Recent evidences have highlighted the implication of Store-Operated Calcium Channels (SOCC) in intracellular calcium homeostasis in coronary artery. However, little is known about the role of SOCC in the regulation of coronary vascular tone under I/R.The aim of this study was to evaluate the role of SOCC and l-type Ca2+ channels (LTCC) in coronary artery vasoconstriction originated by ET-1 in I/R. We used Left Anterior Descendent coronary artery (LAD) rings, isolated from Wistar rats, to study the contractility and intracellular Ca2+ concentration ([Ca2+]i) under a simulated I/R protocol. We observed that responses to high-KCL induced depolarization and caffeine-induced Ca2+ release are attenuated in coronary artery under I/R. Furthermore, ET-1 addition in ischemia promotes transient and small rise of [Ca2+]i and coronary vascular tone. Meanwhile, these effects are significantly potentiated during reperfusion. The resulting ET-1-induced vasoconstrictions and [Ca2+]i increase were abolished by; GSK-7975A and gadolinium, inhibitors of SOCC; and nifedipine a widely used inhibitor of LTCC. Interestingly, using in situ Proximity Ligation Assay (PLA) in isolated coronary smooth muscle cells we found significant colocalization of LTCC CaV1.2 isoform with Orai1, the pore forming subunit of SOCC, and TRPC1 under I/R.Our data suggest that hypercontraction of coronary artery induced by ET-1 after I/R involves the co-activation of LTCC and SOCC, which colocalize significantly in the sarcolemma of coronary smooth muscle cells.

Differential expression of CRAC channel in alloxan induced Diabetic BALB/c mice

Objectives: CRAC (Calcium Release Activated Calcium) channel is one of the most important channels regulating calcium influx and has been involved in many autoimmune diseases. The contribution of CRAC channel in the pathogenesis of Type 1 Diabetes (T1D) has not been described much. Thus, we aimed to study the expression of CRAC channel and inflammatory cytokines like IL-1β (Interleukin -1β) and TNF-α (Tumor Necrosis Factor-α) in the spleen-derived cytotoxic T cells, Bone marrow monocytes (BMM) and macrophages differentiated from BMM in the alloxan induced T1D mice.Materials and methods: BALB/c mice treated with alloxan and vehicle control for 12 and 24 h. Spleen derived T cells; Bone marrow derived monocytes were isolated from the control and diabetic BALB/c mice as well as macrophages differentiated from the control and diabetic BMM.Results: We observed increased expression of CRAC channel components like STIM1 (Stromal Interaction Molecule), ORAI1 and ORAI2 and inflammatory cytokines like IL-1β and TNF-α in the spleen derived cytotoxic T cells and Macrophages differentiated from BMM as well as the downregulated expression of the same and CRAC channel in BMM of 12 and 24 h alloxan induced BALB/c mice.Conclusions: This study suggests that differential expression of CRAC channel correlated with the expression of inflammatory cytokines, thus CRAC channel might be responsible for the increased production of inflammatory cytokines in the alloxan induced T1D mice.

Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons.

The striatum plays an important role in linking cortical activity to basal ganglia output. Striatal neurons exhibit spontaneous slow Ca2+ oscillations that result from Ca2+ release from the endoplasmic reticulum (ER) induced by the mGluR5-IP3R signaling cascade. The maximum duration of a single oscillatory event is about 300 s. A major question arises as to how such a long-duration Ca2+ elevation is maintained. Store-operated calcium channels (SOCCs) are one of the calcium (Ca2+)-permeable ion channels. SOCCs are opened by activating the metabotropic glutamate receptor type 5 and inositol 1,4,5-trisphosphate receptor (mGluR5-IP3R) signal transduction cascade and are related to the pathophysiology of several neurological disorders. However, the functions of SOCCs in striatal neurons remain unclear. Here, we show that SOCCs exert a functional role in striatal GABAergic neurons. Depletion of calcium stores from the ER induced large, sustained calcium entry that was blocked by SKF96365, an inhibitor of SOCCs. Moreover, the application of SKF96365 greatly reduced the frequency of slow Ca2+ oscillations. The present results indicate that SOCCs contribute to Ca2+ signaling in striatal GABAergic neurons, including medium spiny projection neurons (MSNs) and GABAergic interneurons, through elevated Ca2+ due to spontaneous slow Ca2+ oscillations.

Abstract C045: RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML

Abstract Background: Store-operated calcium entry (SOCE) through calcium release activated calcium (CRAC) channels contribute to calcium influx in non-excitable cells. Elevation of cytosolic calcium through SOCE mediates an array of cellular responses including metabolism and gene expression, cell growth, and proliferation. Aberrant CRAC activity has been linked to various auto-immune disorders and certain cancers via the NFAT pathway. Calcium signaling via CRAC channels also mediates several physiological processes including cell proliferation and apoptosis. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. The objective of this study was to evaluate the single agent activity and the combination of RP4010 and standard of care drugs in AML models in vitro and in vivo. Methods: Cell growth following incubation with RP4010 (5 μM), Gilteritinib (0.25-0.75 μM), or a combination of both agents was determined in the AML lines: THP-1, HL-60, KG-1, and U-937. Apoptosis was evaluated following incubation of cell lines (THP-1 and U-937) with compounds for 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. For cell cycle, cells were incubated with compounds for 72 h, fixed with 70% ethanol, incubated at 4°C for 3 h, and stained with Propidium Iodide prior to analysis by flow cytometry. AKT and Stat-5 phosphorylation was determined by Western blotting. In vivo efficacy was evaluated in a MV-4-11 mouse xenograft model. Results: Store-operated calcium entry with RP4010 potentiated Gliteritinib activity manifested by a significant (P&amp;lt;0.05) reduction in cell growth when compared to the activity of the individual agents. Similarly, the combination (3 µM RP4010 + 1 or 2 µM Gilteritinib) led to a 13% increase in the number of apoptotic cells besides increasing cells in the G0/G1 phase by 17%. Combining RP4010 (1 µM) and Gilteritinib (0.1 µM) reduced AKT and Stat-5 phosphorylation by 45 and 57%, respectively, when compared to Gilteritinib in the THP-1 cell line. In the MV-4-11 mouse xenograft model, RP4010 when administered orally at 30 mg/kg/BID for 21 days demonstrated significant (P&amp;lt;0.001) anti-tumor activity both as a single agent and in combination with Cytarabine with tumor growth inhibitions of 42 and 68 %, respectively. Both RP4010, as well as the combination of RP4010 + Cytarabine, were well tolerated throughout the study period with no appreciable change in body weight. Conclusions: RP4010, a potent SOCE inhibitor, attenuated AML tumor growth as a single agent besides demonstrating remarkable potentiation in activity of approved/standard of care drugs for treating AML. Findings provide a rationale for use of this molecule in clinical trials involving naïve or relapsed/refractory AML patients. RP4010 is currently being evaluated in a Phase-1 dose-escalation study in patients with relapsed/refractory lymphomas. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C045. doi:10.1158/1535-7163.TARG-19-C045

Increased Confinement and Polydispersity of STIM1 and Orai1 after Ca2+ Store Depletion

STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca2+ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca2+ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca2+ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca2+ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility—measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient—decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca2+ influx, which can facilitate the rapid activation of local Ca2+ signaling pathways and the efficient replenishing of Ca2+ store in the ER in store-operated Ca2+ entry.