Additional house-keeping genes (and expression in T cells, yielding similar results compared with (not shown). mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 Ginkgolide A and to attenuate CRAC channel function. The combined deletion of and abolishes SOCE and strongly impairs T cell function. 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+ release-activated Ca2+ (CRAC) channels mediate Ca2+ influx in many cell types. They are formed by the tetraspanning plasma membrane proteins ORAI1, ORAI2 and ORAI3. These ORAI proteins mediate Ca2+ influx by store-operated Ca2+ entry (SOCE), so named because it depends on the filling state of Ginkgolide A intracellular Ca2+ stores1. Upon cell stimulation through receptors that are linked to phospholipase C and the production of IP3, Ca2+ is released from the endoplasmic reticulum (ER) via the opening of IP3 receptors. The reduction in the ER Ca2+ concentration is followed by activation of two transmembrane proteins located in the ER membrane, stromal interaction molecule 1 (STIM1) and STIM2 (ref. 1). Dissociation of Ca2+ from the EF hand domains of STIM1 and STIM2 results in conformational changes that enable them to bind and open CRAC channels in the plasma membrane1. CRAC channels are hexameric complexes composed of individual or potentially multiple ORAI homologues2. StructureCfunction analyses of Orai channels as well as human and mouse ORAI1 have shown that they constitute the pore of the CRAC channel2,3,4. The transmembrane domains are highly conserved between all three ORAI homologues. The first transmembrane domain of ORAI1 lines the channel pore and contains Ginkgolide A a glutamate residue that constitutes a high-affinity Ca2+ binding site and confers strong Ca2+ selectivity to the CRAC channel2,3,5,6,7. All three ORAI homologues can function as Ca2+ channels when overexpressed8,9. The properties of ectopically expressed ORAI1, ORAI2 and ORAI3 channels are similar to those of endogenous CRAC channels10,11, including activation by Ca2+ store depletion, high Ca2+ selectivity, inward rectification and Ca2+-dependent inactivation (CDI)1. However, Rabbit Polyclonal to PDXDC1 the three ORAI homologues differ in some of their channel properties, including fast and slow CDI and their sensitivity to pharmacological inhibitors such as 2-aminoethoxydiphenyl borate8,9. CRAC currents (are particularly high in immune cells and those of are high in the brain, lung, spleen and small intestine, whereas mRNA is abundant in many solid organs1,13,15,16. Expression of is also reported in platelets, melanocytes, B cells, dendritic cells, macrophages and mast cells13. ORAI1 is the best-characterized ORAI homologue in terms of its physiological functions, whereas less is known about ORAI2 and ORAI3. Patients with null or loss-of-function mutations in present with a complex disease syndrome, CRAC channelopathy, which is characterized by immunodeficiency, autoimmunity, muscular hypotonia and ectodermal dysplasia because ORAI1 has critical functions in T cells, skeletal muscle cells, dental enamel-forming cells and eccrine sweat glands17,18,19. ORAI1 is of particular importance for T cell function as emphasized by the lack of CRAC currents and SOCE in T cells of patients with null or loss-of-function mutations in (refs 17, 20, 21). The mutations cause a severe combined immunodeficiency-like disease characterized by impaired T cell proliferation, reduced cytokine production, abolished antibody responses and severe viral and bacterial infections17,18. In mice, the Ginkgolide A dependence of CRAC channel function on ORAI1 appears to be less pronounced as naive T cells from mice and knock-in mice (that express a non-functional ORAI1 p.R93W protein) have residual SOCE22,23,24, reduced but not abolished cytokine production and the ability (upon differentiation into proinflammatory T helper 1 (TH1) and TH17 cells) to cause experimental autoimmune encephalomyelitis25,26. These findings suggest that residual SOCE and T.