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

摘要

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, Stim^, and Orai^-, that together form the molecular basis for CRAC channel activity^, . 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)^{, ,} , and Orai embodies the pore of the PM calcium channel^-. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, 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 basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca²⁺ store depletion or clustering of Orai into punctae yielded predominantly 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 a tetramer that constitutes the Ca²⁺-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.