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

Abstract

The mechanism underlying activation of Ca2+ release-activated Ca2+ channels, in response to depletion of intracellular Ca2+ stores, has generated much interest. Recently, it was shown that Stim forms the relay between the Ca2+ store and the plasma membrane, where it activates the ion-channel forming protein Orai. Here, Penna et al. show that Orai is a dimer at rest and a tetramer when activated by Stim, raising the interesting possibility that dimerization is the trigger for channel activation. 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, Stim1,2 and Orai3,4,5, 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 membrane1,8,9, and Orai embodies the pore of the plasma membrane calcium channel10,11,12. A close interaction between Stim and Orai, identified by co-immunoprecipitation12 and by Förster resonance energy transfer13, 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.