The CRAC channel consists of a tetramer formed by Stiminduced dimerization of Orai dimers. Nature 456

Ca2+ release-activated Ca2+ (CRAC) channels underlie sustained Ca2+ signaling in lymphocytes and numerous other cells following Ca2+ liberation from the endoplasmic reticulum (ER). RNAi screening approaches identified two proteins, Stim1, 2 and Orai3-5, that together form the molecular basis for CRAC channel activity6, 7. Stim senses depletion of the ER Ca2+ store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane (PM)1, 8, 9, and Orai embodies the pore of the PM calcium channel10-12. A close interaction between Stim and Orai, identified by co-immunoprecipitation12 and by Förster resonance energy transfer13, is involved in opening the Ca2+ channel formed by Orai subunits. Most ion channels are multimers of poreforming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the PM. Here we show by biochemical analysis after cross-linking in cell lysates and in intact cells, and by non-denaturing gel electrophoresis without cross-linking that Orai is predominantly a dimer in the PM under resting conditions. Moreover, single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent again with a dimeric basa