Identification of critical players in store-operated calcium entry

(a) The plasma membrane Ca²⁺ channels responsible for calcium entry into T cells had been well-characterized electrophysiologically as “store-operated Ca²⁺ entry” or “CRAC (Ca²⁺ release-activated Ca²⁺)” channels that opened when ER Ca²⁺ stores were depleted as a result of inositol trisphosphate (IP3) binding to IP3 receptors, but their molecular identity was unknown. Taking advantage of our previous finding that T cells from two patients with an autosomal recessive immunodeficiency disease showed complete loss of CRAC channel function (PMID 16147976, 11276202), we used a creative combination of positional cloning and a whole-genome RNA interference (RNAi) screen for regulators of NFAT nuclear translocation in Drosophila cells. This effort identified human ORAI1 as the long-sought pore subunit of the CRAC channel and showed that it was inactivated by a single point mutation in the patients’ cells ([5, 6], collaboration with Dr. Patrick Hogan). Our discovery opened a new field, since ORAI channels are components of a critical Ca²⁺ entry pathway that operates in all non-excitable cells. (b) ORAI channels are gated by two ER transmembrane proteins, STIM1 and STIM2. We generated mice conditionally lacking both STIM proteins, and showed that deletion of STIM1 in T cells abrogated store-operated Ca²⁺ entry whereas deletion of both STIM1 and STIM2 blocked the development and function of T regulatory cells (Tregs) [7] and other “agonist-selected” T cells (PMID 23499491). The mice have been made freely available to the research community. (c) To identify new modulators of STIM-ORAI signalling, we performed a second whole-genome RNAi screen in mammalian cells, which identified septins [8] and TMEM110 (PMID: 26644574) as important regulators of efficient STIM1–ORAI1 communication. Our review on STIM-ORAI signalling: PMID: 20307213 5. Feske S*, Gwack Y*, Prakriya M, Srikanth S, Puppel S-H, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. A mutation in Orai1 causes immune deficiency by abrogating store-operated Ca²⁺ entry and CRAC channel function. Nature 2006; 441: 179-185. 6. Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG. Orai1 is an essential pore subunit subunit of the CRAC channel. Nature 2006; 443: 230-233. 7. Oh-hora M, Yamashita M, Hogan PG, Sharma S, Lamperti E, Chung W, Prakriya M, Feske S, Rao A.  Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nature Immunol 2008; 9: 432-443. PMC2737533 8. Sharma S, Quintana A, Findlay GM, Mettlen M, Baust B, Jain M, Nilsson R, Rao A, Hogan PG. A genome-wide screen for NFAT activation identifies septins as essential modulators of store-operated Ca²⁺ entry.  Nature 2013; 499: 238-242. PMC3846693