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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.

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

Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca 2+ entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8 + effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1β and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca 2+ influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.