Store-operated Ca2+ entry through calcium release-activated calcium channels is the chief mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell-specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells.

ORAI1 is a pore subunit of the store-operated Ca 2؉ release-activated Ca 2؉ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1 ؊/؊ mice. Orai1 ؊/؊ mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1 ؊/؊ mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1 ؊/؊ mice, but B cells showed a substantial decrease in Ca 2؉ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1 ؊/؊ T cells showed substantial reductions in store-operated Ca 2؉ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.Ca 2ϩ is a universal second messenger that regulates a multitude of cellular functions, including secretion, muscle contraction, ion channel function, and gene expression (5). In many nonexcitable cells, Ca 2ϩ influx occurs through "storeoperated" Ca 2ϩ channels which open in response to depletion of endoplasmic reticulum (ER) Ca 2ϩ stores (40). Physiologically, this occurs when ligand binds to receptors, such as G protein-coupled receptors, immunoreceptors, and receptor tyrosine kinases, that are coupled to the activation of phospholipase C. The resulting production of inositol trisphosphate leads to efflux of Ca 2ϩ from the ER through inositol trisphosphate receptors and decreased Ca 2ϩ concentration in the ER lumen. This decrease directly regulates the opening of storeoperated Ca 2ϩ channels in the plasma membrane (26). In lymphocytes and other immune system cells, the major route of Ca 2ϩ influx is through store-operated Ca 2ϩ releaseactivated Ca 2ϩ (CRAC) channels. CRAC currents (I CRAC ) were first identified in T cells and mast cells (20,21,27,53), and Ca 2ϩ influx through CRAC channels is known to be essential for T-cell activation (8, 25). Mutant Jurkat tumor T-cell lines lacking functional CRAC channels cannot be activated properly (7); moreover, T cells obtained from three independent families of patients with hereditary severe combined immunodeficiency (SCID) were shown to be severely deficient in store-operated Ca 2ϩ entry and the CRAC channel current, I CRAC (10,13,23,36). T-cell responses, particularly proliferation and cytokine production in vitro in response to T-cell receptor stimulation, were strongly impaired in patients from two of these families, explaining their SC...

Ca2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca 2+ sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca 2+ and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity 1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2+ selectivity and are instrumental in generating sustained [Ca2+]i elevations necessary for gene expression and effector function in many eukaryotic cells 2. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels were not Ca2+-selective, their Ca2+ selectivity was dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity also occurred in wild-type (WT) Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled.

ORAI1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, which is responsible for store-operated Ca2+ entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1−/− mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1KI/KI) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1KI/KI mice die neonatally, but Orai1KI/KI fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1KI/KI mice display severely impaired store-operated Ca2+ entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4+ and CD8+ T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1KI/KI mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1KI/KI mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.