Calcium signaling via Orai1 is essential for induction of the nuclear orphan receptor pathway to drive Th17 differentiation.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1).

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

ORAI1 deficiency impairs activated T cell death and enhances T cell survival.

ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.

The third transmembrane segment of orai1 protein modulates Ca2+ release-activated Ca2+ (CRAC) channel gating and permeation properties.

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 µm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.