1. Department of Physiology and Biophysics,
2. Department of Neurobiology and Behavior, and,
3. Center for Immunology, University of California Irvine, California 92697-4561, USA

Correspondence to: Michael D. Cahalan^1,3 Correspondence and requests for materials should be addressed to M.D.C. (Email: mcahalan@uci.edu).

Abstract

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, 4, 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, 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.

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