alpha-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

作者：Li Peiyao; Miao Yong; Dani Adish; Vig Monika

来源：Molecular Biology of the Cell, 2016, 27(16): 2542-2553.

DOI：10.1091/mbc.E16-03-0163

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and alpha-SNAP is necessary for its function. Here we show that alpha-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. 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. Remarkably, alpha-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in alpha-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that alpha-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of alpha-SNAP with Stim1 and Orai1, and alpha-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, alpha-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of alpha-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.

• 出版日期2016-8-15