ORAI1 Research Articles

The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+ entry

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+ nanodomain, which forms when Ca2+ channels open. Ca2+ nanodomains arising from store-operated Orai1 Ca2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.

Store‐operated calcium entry mediated high glucose‐induced podocyte injury and mitochondrial impairment

Purpose Diabetic Nephropathy is one of the major complications of diabetes mellitus. Hyperglycemia is a known initiator of diabetes mellitus. Accumulating evidence suggests that podocyte injury is tightly associated with the onset and progression of diabetic nephropathy. However, the mechanisms underlying podocyte injury induced by hyperglycemia or high glucose (HG) is poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in the settings of diabetes is not known. The aim of the present study was to examine if SOCE mediated HG-induced podocyte injury by impairing mitochondrial function. Methods All experiments were carried out in cultured immortalized human podocytes. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and nephrin (a podocyte specific protein). Calcium imaging was used to analyze SOCE. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP). Results HG (25mM) significantly increased podocyte Orai1 protein abundance for time periods ranging from 2 to 12 hours. This HG effect was dose dependent. Consistently, Ca2+ imaging experiments showed that HG treatment (25 mM for 12 hours) significantly enhanced podocyte SOCE. Furthermore, the abundance of nephrin protein decreased in podocytes after exposure to HG, indicating podocyte injury in the presence of ambient HG. However, the HG-induced decrease of nephrin was blunted by BTP2 (an SOCE inhibitor, 4 µM). Moreover, HG treatment (25 mM for 24 hours) decreased MMP, indicating damage of mitochondria by HG. The decrease of MMP was prevented by BTP2, suggesting the contribution of SOCE to the detrimental effect of HG treatment. Conclusion The present study suggests that upregulated SOCE contributes to HG-induced podocyte injury, possibly by impairing mitochondrial function.

SET knockdown attenuated phenotype modulation and calcium channel associated markers of airway smooth muscle cells in asthmatic mice.

BackgroundDysfunctional phenotype modulation and calcium channels in airway smooth muscle cells (ASMCs) are important characteristics of airway remodeling in chronic asthma. However, the mechanisms underlying these pathological processes remain unclear. SET (I2PP2A, inhibitor-2 of protein phosphatase 2A) has many significant functions and is involved in various physiological and pathological processes. This study aimed to determine the function of SET in chronic asthma.MethodsBALB/c mice were sensitized by ovalbumin injection and repeated inhalation of ovalbumin. The Penh value was measured using the Buxco whole body plethysmography system. A short hairpin RNA of the SET gene was designed and transfected into ASMCs derived from asthmatic mice. Flow cytometry of Annexin-V/propidium iodide staining was used for evaluating cell apoptosis. Western blot was adopted to measure the expression levels of ASMCs phenotype modulation markers and calcium channel-associated proteins.ResultsThe results showed that shRNA targeting SET significantly decreased the expression of SET, and enhanced the apoptosis of ASMCs. SET knockdown promoted the expression of contractile phenotype markers such as α-SMA (alpha smooth muscle Actin), SM-MHC (smooth muscle Myosin heavy chain), and calponin, and inhibited the expression of synthetic phenotype markers including vimentin and CD44. The expression of the calcium channel-related proteins STIM1 (Stromal interaction molecule 1) and Orai1 were also inhibited after SET knockdown.ConclusionsThese data demonstrated that SET participated in the development of airway dysfunction in asthma, suggesting that the silencing of SET may be a new therapeutic target for the treatment of asthma patients.

STIM1 Regulates Endothelial Calcium Overload and Cytokine Upregulation During Sepsis

BackgroundStromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is now recognized as the main mechanism of the majority of nonexcitable cell calcium influx. Calcium overload is a primary mechanism of endothelial cell injury during systemic inflammatory response and sepsis. Whether STIM1-mediated SOCE plays a role in calcium overload in vascular endothelial cell injury remains unclear. Materials and methodsTo explore the role of STIM1-gated SOCE in vascular endothelial cell calcium overload and inflammation, we established a human septic serum or lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) experimental system and derived ribonucleic acid interference (RNAi)-mediated STIM1, ORAI1 (orai gene [HGNC: 25896 Entrez Gene: 84876] coding protein, ORAI Calcium Release-Activated Calcium Modulator 1), and transient receptor potential channel 1 (TRPC1) (core components of store-operated Ca2+[SOC]) downregulated HUVECs, as well as STIM1 overinduced HUVECs. ResultsOur results show that sepsis serum or LPS stimulation increased STIM1 in HUVECs and increased all cytokines except for VEGF and the inflammatory mediators tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1 in a time-dependent manner. RNAi-mediated knockdown of STIM1 significantly inhibited serum or LPS-induced inflammatory cytokine expression, and STIM1 overexpression in HUVECs promoted LPS-mediated induction of these cytokines. Meanwhile, similar to the blocking effect of the specific SOC inhibitors Gd3+ and La3+ on LPS-induced calcium influx, RNAi-mediated depletion of STIM1 or the SOC proteins TRPC1 and ORAI1 could significantly inhibit serum or LPS-induced extracellular calcium influx, as well as the expression of the inflammatory cytokines tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1. Simultaneous downregulation of the SOCE core units TRPC1 and ORAI1 inhibited LPS-induced calcium influx and cytokine expression, which could not be restored by inducing STIM1. Forced expression of nuclear factor-κB (NF-κB) in HUVECs significantly induced STIM1 expression, whereas RNAi-mediated depletion of NF-κB significantly inhibited STIM1 mRNA levels and significantly reduced the thapsigargin-mediated SOCE calcium influx, which was similar to results with the NF-κB inhibitor wogonin. ConclusionsSeptic serum stimulates the expression of STIM1, cytokines, and inflammatory mediators in HUVECs. STIM1-mediated SOCE is required for Ca2+ influx induced by LPS or septic serum and contributes cytokines and inflammatory mediators in septic serum–stimulated HUVECs. In addition, STIM1-mediated SOCE on Ca2+ influx by septic serum or LPS involves NF-κB signaling.

Junctophilin-4 facilitates inflammatory signalling at plasma membrane-endoplasmic reticulum junctions in sensory neurons.

Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) JPH4 is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. Knockdown of JPH4 impairs endoplasmic reticulum Ca2+ store refill and junctional Ca2+ signalling in sensory neurons. In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. Junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms. Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) form signalling nanodomains in eukaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super-resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin-4 (JPH4), is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER-PM junctional areas and co-clusters with SOCE proteins STIM1 and Orai1 upon ER Ca2+ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca2+ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca2+ signalling in the pain-like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

Novel ORAI1 Mutation Disrupts Channel Trafficking Resulting in Combined Immunodeficiency

Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient’s lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.

Role of Orai 1-mediated store-operated calcium entry in the immune function of CD4+ T cells in septic mice

Objective: To investigate the role of Orai1-mediated store-operated calcium entry in the immune damage of CD4+ T cells in septic mice. Methods: Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, and 5 days after operation. Spleen samples were harvested at given intervals. Splenic CD4+ T cells were selected by immunomagnetic beads and the expression of Orai1 protein was detected by western blotting, the storage operated calcium entry (SOCE) was detected by flow cytometry, the apoptosis of CD4+ T cells was detected by flow cytometry, the proliferation of CD4+ T cells was detected by CCK-8, and the IFN-γ and IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). Then the expression of Orai1 protein was regulated to further detect the SOCE and immune function of splenic CD4+ T cells in mice. The experiment was divided into 4 groups, sham group, CLP3 group, Orai1 down group (Orai1-down group) and Orai1 up regulation group (Orai1-up group). Results: The relative expression of Orai1 protein in splenic CD4+ T cells in sham group was 1.03±0.16. Compared with sham group, Orai1 protein levels in CLP Group were all significantly lower (F=19.64, P=0.000 5). The increased value of splenic CD4+ T cells fluorescence intensity in sham group was 494±41. Compared with sham group, the levels of SOCE in CLP Group were all lower (F=30.01, P=0.001). The ratio of early and late apoptosis of CD4+ T cells in sham group was 8.7%±1.5%. Compared with sham group, the early and late apoptosis rates of CLP Group were significantly higher (F=32.29, P=0.000 1). The OD of sham group was 0.81±0.10 at 450 nm. Compared with sham group, the proliferation ability of splenic CD4+ T cells in CLP Group were significantly decreased (F=7.26, P=0.001 8). Compared with sham group, the secretion of IFN-γ and IL-4 by CD4+ T cells and the ratio of IFN-γ/IL-4 in CLP Group were all significantly decreased (F=19.690, 6.183, 11.230, all P<0.05). Compared with CLP3 group, the increased value of fluorescence intensity of CD4+ T cells was significantly decreased, the early and late apoptosis ratio of CD4+ T cells was significantly increased, the OD450 nm value of CD4+ T cells was decreased, the multiplication capacity of splenic CD4+ T cells were decreased, the level of IFN-γ and IL-4 secreted by T cells were decreased, and the value of IFN-γ/IL-4 in orai1-down group was decreased (t=4.819, 7.952, 2.988, 28.760, 3.140, 7.670, all P<0.05). However, Orail-up group showed the opposite trend. Conclusion: Orai1-mediated store-operated calcium entry can alleviate the immune dysfunction of CD4+ T cells in septic mice.

The Role of S-Acylation in the Regulation of Store-Operated Calcium Entry

Store-operated calcium entry (SOCE) is critical for T cell-mediated immunity, specifically, for T cell activation, clonal expansion, and differentiation. In T cells, SOCE is facilitated by the plasma membrane (PM) localized protein Orai1 and the endoplasmic reticulum (ER) membrane localized protein STIM1. Upon T cell receptor activation, ER calcium stores are depleted, leading to STIM1 activation, which undergoes a conformational change, allowing it to interact with Orai1 at ER/PM junctions. This interaction enables Orai1 gating, leading to calcium entry into the cell. Although the importance of Orai1 and STIM1 in SOCE has been established, it is still unclear what mechanisms regulate the formation of Orai1/STIM1 complexes at ER/PM junctions. We have found that, among several other regulatory T cell proteins, both Orai1 and STIM1 are S-acylated. S-acylation is the post-translational lipidation of a cysteine residue via a labile thioester bond. Using biochemical, electrophysiological, and advanced imaging approaches, we have found that S-acylation of Orai1 and STIM1 is critical for their function. Using acyl-biotin exchange, we determined not only are Orai1 and STIM1 S-acylated, but also the S-acylation of Orai1 is rapid, transient, and calcium-dependent. To further study the importance of S-acylation on SOCE, we used whole cell recording to show that the S-acylation of both Orai1 and STIM1 is important for proper calcium channel currents. Lastly, after performing total internal reflection fluorescence, Förster resonance energy transfer, and Fura-2 calcium imaging, we determined acylation-deficient Orai1 significantly decreases SOCE and does not efficiently interact with STIM1 to form functional calcium channels. This work describes a previously unknown signaling pathway that regulates SOCE in T cells, and contributes to T cell-mediated immunity.

Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation.

The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation.

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

Expression of ORAI1 and STIM1 genes in blood of patients with pulmonary tuberculosis.

This study aimed to detect the expression level of ORAI1 and STIM1 genes in blood of patients with bilateral pulmonary tuberculosis (TB) in comparison with the control group. Both genes encode proteins providing store-operated Ca2+ entry (SOCE) into the cells, including immune cells, to activate transcriptional factors for producing cytokines and inflammation-restricting proteins. The study included 45 patients with confirmed TB, aged 20 to 86, and 35 volunteers, aged from 21 to 73, without active TB infection. The expression of ORAI1 and STIM1 genes in blood was performed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the referent gene. Inflammation was assessed by levels of interferon γ (IFN-γ) and interleukin 18 (IL-18) in serum (ELISA method). The results showed lower expression of ORAI1 in blood and higher levels of IFN-γ and IL-18 in serum of TB patients than that of the control group and no differences in expression of the STIM1 gene. It indicates some impairment in the SOCE mechanism of immune cells, which is associated with TB.

Low intensity repetitive magnetic stimulation reduces expression of genes related to inflammation and calcium signalling in cultured mouse cortical astrocytes

Repetitive transcranial magnetic stimulation (rTMS) is a form of non-invasive brain stimulation frequently used to induce neuroplasticity in the brain. Even at low intensities, rTMS has been shown to modulate aspects of neuronal plasticity such as motor learning and structural reorganisation of neural tissue. However, the impact of low intensity rTMS on glial cells such as astrocytes remains largely unknown. This study investigated changes in RNA (qPCR array: 125 selected genes) and protein levels (immunofluorescence) in cultured mouse astrocytes following a single session of low intensity repetitive magnetic stimulation (LI-rMS – 18 mT). Purified neonatal cortical astrocyte cultures were stimulated with either 1Hz (600 pulses), 10Hz (600 or 6000 pulses) or sham (0 pulses) LI-rMS, followed by RNA extraction at 5 h post-stimulation, or fixation at either 5 or 24-h post-stimulation. LI-rMS resulted in a two-to-four-fold downregulation of mRNA transcripts related to calcium signalling (Stim1 and Orai3), inflammatory molecules (Icam1) and neural plasticity (Ncam1). 10Hz reduced expression of Stim1, Orai3, Kcnmb4, and Ncam1 mRNA, whereas 1Hz reduced expression of Icam1 mRNA and signalling-related genes. Protein levels followed a similar pattern for 10Hz rMS, with a significant reduction of STIM1, ORAI3, KCNMB4, and NCAM1 protein compared to sham, but 1Hz increased STIM1 and ORAI3 protein levels relative to sham. These findings demonstrate the ability of 1Hz and 10Hz LI-rMS to modulate specific aspects of astrocytic phenotype, potentially contributing to the known effects of low intensity rTMS on excitability and neuroplasticity.

Store-operated calcium entry: Pivotal roles in renal physiology and pathophysiology.

Research conducted over the last two decades has dramatically advanced the understanding of store-operated calcium channels (SOCC) and their impact on renal function. Kidneys contain many types of cells, including those specialized for glomerular filtration (fenestrated capillary endothelium, podocytes), water and solute transport (tubular epithelium), and regulation of glomerular filtration and renal blood flow (vascular smooth muscle cells, mesangial cells). The highly integrated function of these myriad cells effects renal control of blood pressure, extracellular fluid volume and osmolality, electrolyte balance, and acid-base homeostasis. Many of these cells are regulated by Ca2+ signaling. Recent evidence demonstrates that SOCCs are major Ca2+ entry portals in several renal cell types. SOCC is activated by depletion of Ca2+ stores in the sarco/endoplasmic reticulum, which communicates with plasma membrane SOCC via the Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Orai1 is recognized as the main pore-forming subunit of SOCC in the plasma membrane. Orai proteins alone can form highly Ca2+ selective SOCC channels. Also, members of the Transient Receptor Potential Canonical (TRPC) channel family are proposed to form heteromeric complexes with Orai1 subunits, forming SOCC with low Ca2+ selectivity. Recently, Ca2+ entry through SOCC, known as store-operated Ca2+ entry (SOCE), was identified in glomerular mesangial cells, tubular epithelium, and renovascular smooth muscle cells. The physiological and pathological relevance and the characterization of SOCC complexes in those cells are still unclear. In this review, we summarize the current knowledge of SOCC and their roles in renal glomerular, tubular and vascular cells, including studies from our laboratory, emphasizing SOCE regulation of fibrotic protein deposition. Understanding the diverse roles of SOCE in different renal cell types is essential, as SOCC and its signaling pathways are emerging targets for treatment of SOCE-related diseases.

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

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

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

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

Orai-1 and Orai-2 regulate oral cancer cell migration and colonisation by suppressing Akt/mTOR/NF-κB signalling

Despite remarkable progress in understanding and treating oral cancer (OC), it still remains one of the life-threatening diseases and predominant cancers in the world. Therefore, deciphering the molecular mechanisms of this disease would help us to develop highly efficacious therapies. Multiple lines of evidence suggest that calcium and its dysregulation play significant role in the development of various cancers. As an adaptation of survival mechanism, upon depletion of ER calcium stores, store-operated calcium entry (SOCE) has been induced via SOCE channels (SOCC) in various mammalian cells. SOCC are regulated by Orai-1, Orai-2 and Orai-3 located on plasma membrane and two calcium-sensing ER membrane proteins known as stromal interaction molecules (STIM-1 and STIM-2). Hence, the present study was aimed at analysing the role of Orai-1 and Orai-2 in oral cancer and the underlying mechanism. Our results suggest that both Orai-1 and Orai-2 proteins were overexpressed in oral cancer tissues and cell lines (SAS) compared to normal epithelial tissues and cell lines respectively. In addition, silencing of Orai-1 and Orai-2 via chemical SOCE inhibitors and siRNAs inhibited calcium uptake and suppressed oral cancer cell proliferation, colony formation and migration. Furthermore, silencing of Orai-1 and Orai-2 inhibited Akt/mTOR/NF-κB pathway in oral cancer cells. Interestingly, tobacco carcinogen NNN and synthetic carcinogen 4-NQO, enhanced the expression of Orai-1 and Orai-2 in SAS cells. Therefore, we conclude that Orai-1 and Orai-2 have significant role in oral cancer and can be further explored to develop novel therapies for the treatment of this disease.

17β‐Estradiol via Orai1 activates calcium mobilization to induce cell proliferation in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal estrogen-sensitive gynecological cancer. Studies have reported that estrogen induces rapid cellular calcium mobilization in cells and can determine the fate of a cell. We found that estrogen increased the calcium release-activated calcium channel modulator 1 (Orai1) protein expression levels in SK-OV-3 cells. However, to date, there has been no research on the functional relationship and molecular mechanism of estrogen-regulating Orai1 during EOC development. In our study, Orai1 had a high expression level in high-grade serous ovarian tumor tissues and SK-OV-3 cells. Estrogen promoted cell proliferation and migration while inhibiting cell apoptosis in SK-OV-3 cells. Orai1 silencing suppressed estrogen-induced cell migration and proliferation. Overexpression of Orai1, however, enhanced the ability of 17β-estradiol (E2) to exert its function. Estrogen induced rapid calcium influx in SK-OV-3 cells. Knockdown of Orai1 in SK-OV-3 cells blocked E2-induced stored-operated Ca2+ influx. The messenger RNA expression of caspase 3, matrix metallopeptidase 1, and cyclin-dependent kinase 6 were regulated via Orai1 under E2 treatment. Our results suggest that estrogen, by regulating Orai1, induced calcium influx to determine cell fate.

Calcium entry units (CEUs): perspectives in skeletal muscle function and disease

In the last decades the term Store-operated Ca2+ entry (SOCE) has been used in the scientific literature to describe an ubiquitous cellular mechanism that allows recovery of calcium (Ca2+) from the extracellular space. SOCE is triggered by a reduction of Ca2+ content (i.e. depletion) in intracellular stores, i.e. endoplasmic or sarcoplasmic reticulum (ER and SR). In skeletal muscle the mechanism is primarily mediated by a physical interaction between stromal interaction molecule-1 (STIM1), a Ca2+ sensor located in the SR membrane, and ORAI1, a Ca2+-permeable channel of external membranes, located in transverse tubules (TTs), the invaginations of the plasma membrane (PM) deputed to propagation of action potentials. It is generally accepted that in skeletal muscle SOCE is important to limit muscle fatigue during repetitive stimulation. We recently discovered that exercise promotes the assembly of new intracellular junctions that contains colocalized STIM1 and ORAI1, and that the presence of these new junctions increases Ca2+ entry via ORAI1, while improving fatigue resistance during repetitive stimulation. Based on these findings we named these new junctions Ca2+ Entry Units (CEUs). CEUs are dynamic organelles that assemble during muscle activity and disassemble during recovery thanks to the plasticity of the SR (containing STIM1) and the elongation/retraction of TTs (bearing ORAI1). Interestingly, similar structures described as SR stacks were previously reported in different mouse models carrying mutations in proteins involved in Ca2+ handling (calsequestrin-null mice; triadin and junctin null mice, etc.) or associated to microtubules (MAP6 knockout mice). Mutations in Stim1 and Orai1 (and calsequestrin-1) genes have been associated to tubular aggregate myopathy (TAM), a muscular disease characterized by: (a) muscle pain, cramping, or weakness that begins in childhood and worsens over time, and (b) the presence of large accumulations of ordered SR tubes (tubular aggregates, TAs) that do not contain myofibrils, mitochondria, nor TTs. Interestingly, TAs are also present in fast twitch muscle fibers of ageing mice. Several important issues remain un-answered: (a) the molecular mechanisms and signals that trigger the remodeling of membranes and the functional activation of SOCE during exercise are unclear; and (b) how dysfunctional SOCE and/or mutations in Stim1, Orai1 and calsequestrin (Casq1) genes lead to the formation of tubular aggregates (TAs) in aging and disease deserve investigation.

No social distancing between ORAI channels

ORAI1 is established as an essential component of Ca2+ release-activated Ca2+ (CRAC) channel which mediates store-operated Ca2+ entry (SOCE). However, the contributions of ORAI2 and ORAI3 to SOCE are not understood. We highlight a recent study which shows that ORAI proteins form heteromeric channels which tune SOCE over a range of stimulus intensities.

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.