Stim-regulated Assembly And Stoichiometry Of The CRAC Channel Subunit Orai

Recent RNAi screens have identified Stim and Orai as critical components of the Ca2+ release-activated Ca2+ (CRAC) channel. Stim senses depletion of the Endoplasmic Reticulum (ER) Ca2+ store, translocates from the ER to junctions adjacent to the plasma membrane (PM), and activates Orai pore-forming channel subunits in the PM to open the CRAC channel. The Orai oligomerization interface was investigated by co-immunoprecipitation of N-and/or C-terminal Orai deletion mutants and expressed Orai N- and C-terminal fragments. The transmembrane core domain plays a predominant role in subunit assembly; a weaker interaction interface was identified at the N-terminal region. We analyzed the quaternary structure of the Orai subunit and showed by cross-linking, and by non-denaturing gel electrophoresis that Orai is predominantly a dimer under resting conditions with or without co-expression of Stim. Single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent 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 Orai stoichiometry of the active CRAC channel. Interaction of the Orai C-terminal coiled-coil domain (as shown by structure-disruptive mutations) 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 and may reveal a new channel gating mechanism. New data will be presented on the Stim-Orai stoichiometry and activation mechanism.