The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers

Ca2+-release- activated Ca2+ (CRAC) channels underlie sustained Ca2+ signalling in lymphocytes and numerous other cells after Ca2+ liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim(1,2) and Orai(3-5), that together form the molecular basis for CRAC channel activity(6,7). 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(1,8,9), and Orai embodies the pore of the plasma membrane calcium channel(10-12). A close interaction between Stim and Orai, identified by co-immunoprecipitation(12) and by Forster resonance energy transfer(13), is involved in the opening of the Ca2+ channel formed by Orai subunits. Most ion channels are multimers of pore- forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross- linking in cell lysates and intact cells and by using non- denaturing gel electrophoresis without cross- linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single- molecule imaging of green fluorescent protein (GFP)- tagged Orai expressed in Xenopus oocytes showed predominantly two- step photobleaching, again consistent with a dimeric basal state. In contrast, co- expression of GFP- tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded mostly four- step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca2+-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.