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T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

T cell activation and function depend on Ca2+signals mediated by store-operated Ca2+entry (SOCE) through Ca2+release–activated Ca2+(CRAC) channels formed by ORAI1 proteins. We here investigated how SOCE controls T cell function in pulmonary inflammation during a T helper 1 (TH1) cell–mediated response to influenza A virus (IAV) infection and TH2 cell–mediated allergic airway inflammation. T cell–specific deletion ofOrai1did not exacerbate pulmonary inflammation and viral burdens following IAV infection but protected mice from house dust mite–induced allergic airway inflammation. ORAI1 controlled the expression of genes including p53 and E2F transcription factors that regulate the cell cycle in TH2 cells in response to allergen stimulation and the expression of transcription factors and cytokines that regulate TH2 cell function. Systemic application of a CRAC channel blocker suppressed allergic airway inflammation without compromising immunity to IAV infection, suggesting that inhibition of SOCE is a potential treatment for allergic airway disease.

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in expression of Orai isoforms in response to B cell activation. We show that Orai3 and Orai1 are essential components of native CRAC channels in B cells and are critical for primary B cell proliferation and survival. The combined loss of Orai1 and Orai3 strongly impairs SOCE, nuclear factor for activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of B cells in response to antigenic stimulation. Our results clarify the molecular composition and cellular functions of SOCE in B lymphocytes.

T cells are an essential component of the immune system that provide antigen-specific acute and long lasting immune responses to infections and tumors, ascertain the maintenance of immunological tolerance and, on the flipside, mediate autoimmunity in a variety of diseases. The activation of T cells through antigen recognition by the T cell receptor (TCR) results in transient and sustained Ca(2+) signals that are shaped by the opening of Ca(2+) channels in the plasma membrane and cellular organelles. The dynamic regulation of intracellular Ca(2+) concentrations controls a variety of T cell functions on the timescale of seconds to days after signal initiation. Among the more recently identified roles of Ca(2+) signaling in T cells is the regulation of metabolic pathways that control the function of many T cell subsets. In this review, we discuss how Ca(2+) regulates several metabolic programs in T cells such as the activation of AMPK and the PI3K-AKT-mTORC1 pathway, aerobic glycolysis, mitochondrial metabolism including tricarboxylic acid (TCA) cycle function and oxidative phosphorylation (OXPHOS), as well as lipid metabolism.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3−/− mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3−/− mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3−/− mice. Moreover, Orai3−/− mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies. Mutations in the RNA-binding proteins Roquin-1 or Regnase-1 cause systemic autoimmunity. Heissmeyer and colleagues show that Roquin-1 and Regnase-1 physically interact and thereby regulate CD4+ and CD8+ T cell metabolism and functionality.

T cell antigen receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent mRNA modification affecting splicing, translation and stability of transcripts. Here, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry (SOCE) activity and diminished survival of T cells. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N

ObjectiveInflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses and constitutes a major clinical challenge in need of new treatment modalities to improve patient care. Store-operated Ca2+ entry (SOCE) is the predominant Ca2+ influx pathway in T cells and other immune cells, regulating many of their functional properties. It is currently unknown whether the pharmacologic blockade of SOCE represents a suitable drug-target for IBD treatment.DesignUsing mass and flow cytometry the effects of SOCE inhibition on lamina propria (LP) immune cells of patients with ulcerative colitis (UC) and Crohn’s disease (CD) were investigated. Primary organoid cultures served to study the impact of SOCE inhibition on the function, differentiation and survival of intestinal epithelial cells (IEC). T cell transfer models of colitis were applied to examine how the genetic or pharmacologic ablation of SOCE affects the clinical course of IBD in mice.ResultsWe observed that the LP of IBD patients is characterized by an enrichment of innate lymphoid cells (ILC), CD4+ and CD8+ effector- as well as T regulatory cells producing IL-17 and TNFα. The pharmacologic inhibition of SOCE attenuated the production of pathogenic cytokines including IL-2, IL-4, IL-6, IL-17, TNFα and IFNγ by human colonic T cells and ILC, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, without affecting the viability, differentiation and function of primary IEC. T cell-specific genetic deletion of the SOCE signaling components Orai1, Stim1 or Stim2 revealed that the magnitude of SOCE correlates with the function of T cells and intestinal inflammation in mice. Moreover, the pharmacologic inhibition of SOCE alleviated the clinical course of colitic mice.ConclusionOur data suggest that SOCE inhibition may serve as a new pharmacologic strategy for treating IBD.

TCR stimulation triggers Ca

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination of neurons in the central nervous system (CNS) (1). While the ultimate cause of the disease is unknown, MS is commonly considered to be an autoimmune disorder in which autoreactive CD4+ T cells are activated by an (unknown) autoantigen presented by antigen-presenting cells (APCs) outside the CNS. Once in the CNS, T cells are activated by microglia-derived or CNS-infiltrating APC, resulting in inflammation and myelin damage. The main T cell subsets causing disease in MS are T helper (Th) 1 and Th17 cells, which produce the proinflammatory cytokines interferon gamma (IFN-γ), interleukin (IL)-17A, and granulocyte–macrophage colony-stimulating factor. Studies in MS patients have shown that IL-17A and IFN-γ levels are increased in T cells isolated from CNS lesions and the cerebrospinal fluid (2, 3). A similar role of Th1 and Th17 cells has been found in the experimental autoimmune encephalomyelitis (EAE) rodent model of MS. The proinflammatory function of Th1 and Th17 cells is kept in check by regulatory T (Treg) cells, a subset of CD4+ T cells that express the transcription factor Foxp3 by suppressing effector T cell functions and thus CNS inflammation. Treg cells attenuate the severity of EAE and contribute to recovery from CNS inflammation (4). A report by Othy et al. (5) sheds light on the spatiotemporal interaction of Th17 cells, Treg cells, and APCs and their migration at three different stages of EAE. It also shows how Treg cells modulate Ca2+ signals in Th17 cells in the spinal cord. For their studies, Othy et al. (5) developed Foxp3EGFP IL-17TdT reporter mice to express enhanced green fluorescent protein (EGFP) in Treg cells and TdTomato in Th17 cells, as well as Foxp3EGFP CD11cEYFP reporter mice. Using this fate-mapping approach, the … [↵][1]1To whom correspondence may be addressed. Email: feskes01{at}nyumc.org. [1]: #xref-corresp-1-1

It is widely assumed that inositol trisphosphate (IP3) and ryanodine (Ry) receptors share the same Ca2+ pool in central mammalian neurons. We now demonstrate that in hippocampal CA1 pyramidal neurons IP3- and Ry-receptors are associated with two functionally distinct intracellular Ca2+ stores, respectively. While the IP3-sensitive Ca2+ store refilling requires Orai2 channels, Ry-sensitive Ca2+ store refilling involves voltage-gated Ca2+ channels (VGCCs). Our findings have direct implications for the understanding of function and plasticity in these central mammalian neurons., It is generally assumed, from previous studies in cerebellar Purkinje neurons, that IP3 and Ry receptors share the same intracellular Ca2+ pool. In this study, the authors demonstrate the existence of two functionally distinct Ca2+ stores in mouse CA1 pyramidal neurons by showing that refilling of IP3-sensitive stores relies on Orai2 channels, whereas Ry-sensitive stores involve VGCCs for spontaneous store refilling

Ca2+ release-activated Ca2+ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca2+ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders.

The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca(2+) signals generated by Orai1 activate Ca(2+)-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca(2+) entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca(2+) promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca(2+)](i) increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP(2)-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca(2+) depletion by binding to Orai1 and caused local and global [Ca(2+)](i) increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP(2)-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca(2+) influx to NFAT1 activation.

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca2+ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca2+ influx and cell function in TRPC1/6-deficient (TRPC1/6−/−) and STIM1/2- deficient (STIM1/2ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6−/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6−/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.

Ion channels and transporters move ions across membrane barriers and are essential for a host of cell functions in many organs. They conduct K(+), Na(+) and Cl(−), which are essential for regulating the membrane potential, H(+) to control intra- and extracellular pH and divalent cations such as Ca(2+), Mg(2+) and Zn(2+), which function as second messengers and cofactors for many proteins. Inherited channelopathies due to mutations in ion channels or their accessory proteins cause a variety of diseases in the nervous, cardiovascular and other tissues, but channelopathies that affect immune function are not as well studied. Mutations in ORAI1 and STIM1 genes that encode the Ca(2+) release-activated Ca(2+) (CRAC) channel in immune cells, the Mg(2+) transporter MAGT1 and the Cl(−) channel LRRC8A all cause immunodeficiency with increased susceptibility to infection. Mutations in the Zn(2+) transporters SLC39A4 (ZIP4) and SLC30A2 (ZnT2) result in nutritional Zn(2+) deficiency and immune dysfunction. These channels, however, only represent a fraction of ion channels that regulate immunity as demonstrated by immune dysregulation in channel knockout mice. The immune system itself can cause acquired channelopathies that are associated with a variety of diseases of nervous, cardiovascular and endocrine systems resulting from autoantibodies binding to ion channels. These autoantibodies highlight the therapeutic potential of functional anti-ion channel antibodies that are being developed for the treatment of autoimmune, inflammatory and other diseases.

Background Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), both representing major clinical challenges in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in immune cells regulating many of their functional properties. Conditional knockout mice, in which SOCE signaling components are deleted in T cells, have revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD and it remains elusive, which immune cell subsets are affected by the pharmaceutical blockade of SOCE. Therefore, we here aimed to investigate the effects of the SOCE inhibitor BTP-2 on the function and activation of lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes (LPMC) were isolated from UC and CD patients undergoing colon resection. Cells were ex -vivo stimulated with PMA/Ionomycin in the presence or absence of increasing concentrations of BTP-2, fixed and stored at -80°C until acquisition by mass cytometry. LPMCs were stained using 37 lineage and functional markers targeting B, T, NK or myeloid cells and the resulting flow cytometry standard (FCS) files were analyzed by using cytobank and the R/Bioconductor 9 packages. Additionally, Ca2+ influx measurements were performed in order to assess the impact of SOCE inhibition on the calcium homeostasis of various immune cell subsets. Results Data on B, T, NK, and myeloid cells revealed that treatment with BTP-2 causes a dose-dependent inhibition of calcium influx, demonstrating that BTP-2 is a potent inhibitor of SOCE in human LPMCs and PBMCs. By applying mass cytometry, we investigated how the gradual pharmacologic inhibition of SOCE affects the activation and pro-inflammatory functions of various immune cell subsets obtained from colon specimens of patients with UC and CD, which were characterized by the accumulation of CD4+ effector T cells, IFNγ-producing CD8+ T cells and IL-17-producing innate lymphoid cells (ILCs) compared to non-inflamed specimens. Remarkably, inhibition of SOCE attenuated the production of cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by T cells and ILCs, reduced the activation of B cells and decreased the production of IFNγ by myeloid cells without affecting the viability of primary human epithelial cells. Conclusion Our data reveal for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

Store-operated Ca2+ entry (SOCE) is a conserved mechanism of Ca2+ influx that regulates Ca2+ signaling in many cell types. SOCE is activated by depletion of endoplasmic reticulum (ER) Ca2+ stores in response to physiological agonist stimulation. After it was first postulated by J.W. Putney Jr. in 1986, SOCE has been described in a large number of non-excitable cell types including secretory cells of different exocrine glands. Here we discuss the mechanisms by which SOCE controls salt and fluid secretion in exocrine glands, with a special focus on eccrine sweat glands. In sweat glands, SOCE plays an important, non-redundant role in regulating the function of Ca2+-activated Cl− channels (CaCC), Cl− secretion and sweat production. In the absence of key regulators of SOCE such as the CRAC channel pore subunit ORAI1 and its activator STIM1, the Ca2+-activated chloride channel TMEM16A is inactive and fails to secrete Cl−, resulting in anhidrosis in mice and human patients.

Background Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition. Results Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.

Supplemental Digital Content is available in the text., Background: STIM1 (stromal interaction molecule 1) is a calcium (Ca2+) sensor that regulates cardiac hypertrophy by triggering store-operated Ca2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca2+ load independent of store-operated Ca2+ entry, we hypothesized that it controls electrophysiological function and arrhythmias in the adult heart. Methods: Inducible myocyte-restricted STIM1-KD (STIM1 knockdown) was achieved in adult mice using an αMHC (α-myosin heavy chain)-MerCreMer system. Mechanical and electrophysiological properties were examined using echocardiography in vivo and optical action potential (AP) mapping ex vivo in tamoxifen-induced STIM1flox/flox-Cretg/− (STIM1-KD) and littermate controls for STIM1flox/flox (referred to as STIM1-Ctl) and for Cretg/− without STIM deletion (referred to as Cre-Ctl). Results: STIM1-KD mice (N=23) exhibited poor survival compared with STIM1-Ctl (N=22) and Cre-Ctl (N=11) with >50% mortality after only 8-days of cardiomyocyte-restricted STIM1-KD. STIM1-KD but not STIM1-Ctl or Cre-Ctl hearts exhibited a proclivity for arrhythmic behavior, ranging from frequent ectopy to pacing-induced ventricular tachycardia/ventricular fibrillation (VT/VF). Examination of the electrophysiological substrate revealed decreased conduction velocity and increased AP duration (APD) heterogeneity in STIM1-KD. These features, however, were comparable in VT/VF(+) and VT/VF(−) hearts. We also uncovered a marked increase in the magnitude of APD alternans during rapid pacing, and the emergence of a spatially discordant alternans profile in STIM1-KD hearts. Unlike conduction velocity slowing and APD heterogeneity, the magnitude of APD alternans was greater (by 80%, P

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell‐specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non‐pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell‐mediated inflammatory diseases., Pathogenic Th17 cells have been implicated in autoimmune diseases, while non‐pathogenic Th17 cells provide immunity to fungal pathogens. Patients with mutations in ORAI1 or STIM1 have impaired Ca2+ signaling in immune cells and are more susceptible to infections with fungal pathogens.

Store-operated Ca(2+) entry (SOCE) channels are highly selective Ca(2+) channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca(2+) fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2(−/−) mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca(2+) uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca(2+) influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

Store-operated Ca2+ entry (SOCE) channels are highly selective Ca2+ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca2+ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2−/− mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca2+ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca2+ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells., Regulatory T (Treg) cells are important for maintaining immune homeostasis. Here the authors show that STIM1 and STIM2, which activate the Ca2+ channel ORAI1, are essential for the differentiation of peripheral Treg cells into tissue-resident and follicular Treg cells and their ability to limit autoimmunity in mice.

Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca(2+)) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels. We here show that abolishing STIM1 and Ca(2+) influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells impairs the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS), but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca(2+) influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.

The 16th FASEB Science Research Conference on Ca2+ and Cell Function took place in Lisbon, Portugal, on June 12–17, 2016. Chaired by Stefan Feske (New York University, organizer) and Murali Prakriya (Northwestern University, co-organizer), it brought together over 130 scientists from North America, Europe, and Asia.