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

Abstract

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, Stim1, 2 and Orai3-5, 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 membrane (PM)1, 8, 9, and Orai embodies the pore of the PM calcium channel10-12. A close interaction between Stim and Orai, identified by co-immunoprecipitation12 and by Förster resonance energy transfer13, 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.

STIM protein coupling in the activation of Orai channels

“…Aggregation of Orai1 into m-sized clusters in the PM was recently reported to occur through altered electrostatic interactions of acidic residues in the Orai1 C terminus (33), possibly mediated by the STIMct. Whereas 2-APB causes a massive association between STIMct and Orai1, some constitutive association between STIMct and Orai1 can occur without 2-APB, a result agreeing with 3 recent reports in which STIM1ct was examined (24,30,34). Clearly, a small amount of S1ct is constitutively bound to Orai1 in the membrane (see Fig.…”

“…Clearly, a small amount of S1ct is constitutively bound to Orai1 in the membrane (see Fig. 5 B and F), but there is no evidence of spatial organization of Orai1 without 2-APB, a result also noted by others (30,34). We also observe no changes of 2-APB on endogenous STIM1 puncta formation.…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…The CAD fragment forms a tetramer in solution ), whereas the longer STIM1 cytosolic fragments generally appear as dimers (Ji et al 2008;Penna et al 2008;Muik et al 2009;Yuan et al 2009;Zhou et al 2010a) that may need to unfold to expose the CAD and activate Orai1 (Korzeniowski et al 2010;Muik et al 2011). These findings raise the intriguing possibility that on store depletion, STIM1 undergoes a conformational change that results in formation of a tetramer through interactions among multiple CADs (Covington et al 2010), an idea that remains to be tested.…”

Plasticity in Ca 2+ selectivity of Orai1/Orai3 heteromeric channel

“…Functional Orai channels have been demonstrated to form tetramers (25,26) and are capable of heteromerization. The above described co-expression of Orai1 and Orai3 probably resulted in a repertoire of possible heteromeric but also homomeric channels with functionally overlapping currents.…”