Orai1 subunit stoichiometry of the mammalian CRAC channel pore

Abstract

Agonist-activated Ca2+ entry plays a critical role in Ca 2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca 2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca 2+ release-activated Ca 2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

Functional stoichiometry of the unitary calcium-release-activated calcium channel

“…Conversely, no FRET signal was detected in cells coexpressing Orai1-mKO and four-tandem Orai1-EGFP, presumably because of efficient assembly of a channel pore by the four-tandem constructs alone leaving no spot for a fifth subunit. This latter result is quite analogous to the electrophysiological finding that monomeric dominant negative Orai1 subunits could not join and suppress current in channels formed by four-tandems (19). Our experiments on living cells give the same answers as those in fixed cells and show that fixation, which was necessary to stop the molecular motions in single-molecule TIRF experiments, need not disturb protein stoichiometry.…”

“…Thus, Orai1 proteins associate with each other in vivo (3,17,18), but the stoichiometry for forming a CRAC channel is controversial. A recent study (19) adopted techniques previously used to determine the composition of mammalian potassium channels (20); they recorded macroscopic currents from HEK293 cells coexpressing STIM1 with preassembled tandem multimeric Orai1 constructs and a dominant negative Orai1 E106Q mutant. All of the tandem Orai1 constructs showed small but appropriate CRAC-like currents; however, only cells transfected with Orai1 4 tandems plus STIM1 showed CRAC current that was insensitive to coexpression of a monomeric dominant negative Orai1 mutant.…”

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

“…A tetramer of Orai1 subunits was first proposed by Mignen et al based mainly on the ability of a dominant-negative pore mutant (E106Q) to inhibit currents produced by dimeric or trimeric but not tetrameric Orai1 concatemers (Mignen et al 2008). In cells transfected with monomeric Orai1-GFP and subsequently fixed, quantitation of photobleaching steps of single Orai1-GFP particles also supported a tetrameric stoichiometry (Ji et al 2008), consistent with population brightness analysis of mobile Orai1-GFP after photobleaching in unfixed cells (Madl et al 2010).…”

Calcium signaling in lymphocytes

“…At these late time, STIM1 is inactivated by rebinding of Ca 2+ . Consistent with this hypothesis, ORAI1 exists in the plasma membrane as at least a dimer, and its active form is likely to be a homotetramer [46,50]. Moreover, combined overexpression of ORAI1 and STIM1 leads to marked increases in CRAC current in cells[•49,51,52].…”