Faculty Bibliography

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is critical for lymphocyte function and immune responses. CRAC channels are hexamers of ORAI proteins that form the channel pore, but the contributions of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2 double-deficient mice have impaired T cell-dependent antiviral immune responses, and are protected from T cell-mediated autoimmunity and alloimmunity in models of colitis and graft-versus-host disease. Our study demonstrates that ORAI1 and ORAI2 form heteromeric CRAC channels, in which ORAI2 fine-tunes the magnitude of SOCE to modulate immune responses.

Ca2+ signals were reported to control lipid homeostasis, but the Ca2+ channels and pathways involved are largely unknown. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca2+ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1alpha), and peroxisome proliferator-activated receptor alpha (PPARalpha). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.

Store-operated Ca²⁺entry (SOCE) is a pathway for increasing intracellular Ca²⁺ levels regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca²⁺ channel ORAI1. SOCE-deficient patients suffer from Calcium Release-Activated Calcium (CRAC) channelopathy characterized by immunodeficiency, autoimmunity, myopathy, and anhidrotic ectodermal dysplasia. Several mitochondrial enzymes/complexes depend on Ca²⁺ but the source of Ca²⁺ required for their function are not entirely clear. We recently showed a cell-intrinsic role of SOCE in human mitochondria (Maus M et al. Cell Metab. 2017;25(3):698- 712). MitoView Green showed reduced mitochondrial volume in fibroblasts of patients with ORAI1/STIM1 lossof- function mutations. mtDNA copy numbers and mRNAs expression of selected mitochondrial transcription factors were normal. SDS-PAGE/Western blot analysis showed reduced expression of NADH ubiquinone oxidoreductase subunit-B8, Cytochrome b-c1 complex subunit-2, Cytochrome c oxidase subunit-I, Cytochrome C, Mitochondrial porin and permeability transition pore, etc. Blue native PAGE of isolated mitochondria confirmed reduced expression of CI, CIV and supercomplex CICIII2. SOCE-deficient fibroblasts had reduced mRNA and protein expression of uncoupling protein 2, higher basal mitochondrial membrane potential (MMP) and higher numbers of damaged mitochondria as suggested by increased co-localization of mitochondria and lysosomes and increased MitoKeima reporter activity indicative of lysosomal mitophagy. Oligomycininduced ATP-synthase inhibition revealed decreased electron transport and proton pumping rates measured as MMP hyperpolarization rates and reduced superoxide production assessed by MitoSOX. Maximal O2 consumption rates in SOCE-deficient cells were decreased. Skeletal myocytes had reduced CI and CIV function in 2 out of 3 ORAI1-deficient patients. Gene expression of very long chain acyl-CoA dehydrogenase and long-chain fatty acid transporter carnitine palmitoyltransferase 1B was reduced in patient fibroblasts cultured in either high glucose medium or oleic acid (OA) medium followed by starvation in 2 mM glucose medium. Furthermore, SOCE-deficient fibroblasts were lacking a starvation-induced increase in etomoxir-sensitive mitochondrial respiration in OA medium and showed reduced rates of OA beta-oxidation when cultured in 14C-OA-medium with or without subsequent starvation. Our findings indicate an important new role of SOCE in mitochondrial function

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.

The function of CD4+ T cells is dependent on Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-gamma, and GM-CSF despite only partially reduced Ca2+ influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORgammat. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4+ T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-gamma, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.

Ca2+ release-activated Ca2+ (CRAC) channels mediate a specific form of Ca2+ influx called store-operated Ca2+ entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca2+ levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca2+ homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.