title: α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.
abstract: Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-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, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. 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. 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. Furthermore, α-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 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.
authors.affiliation: Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110.
authors.forename: Peiyao
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110.
authors.forename: Yong
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110 Hope Center for Neurological Disorders, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110 adani@wustl.edu mvig@wustl.edu.
authors.forename: Adish
authors.initials: A
authors.lastname: Dani
authors.affiliation: Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110 adani@wustl.edu mvig@wustl.edu.
authors.forename: Monika
authors.initials: M
authors.lastname: Vig
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D000071740:ORAI1 Protein / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1 / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D000071740:ORAI1 Protein; D015222:Sodium Channels; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D000071737:Stromal Interaction Molecule 1; D002118:Calcium