Store-operated calcium entry (SOCE) via calcium release–activated channels (CRACs) is induced in response to signaling via a variety of cell surface receptors and is crucial for many different cellular processes. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function.
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