title: Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states.
abstract: We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.
authors: Department of Neurobiology and Behavior, Institute for Immunology, University of California, Irvine, CA 92697, USA. ademuro@uci.edu
authors: Angelo
authors: A
authors: Demuro
authors: Aubin
authors: A
authors: Penna
authors: Olga
authors: O
authors: Safrina
authors: Andriy V
authors: AV
authors: Yeromin
authors: Anna
authors: A
authors: Amcheslavsky
authors: Michael D
authors: MD
authors: Cahalan
authors: Ian
authors: I
authors: Parker
mesh_terms: D015153:Blotting, Western; D001896:Boron Compounds; D015220:Calcium Channels / Q000737:chemistry*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D005456:Fluorescent Dyes; D016257:Fura-2; D049452:Green Fluorescent Proteins; D006801:Humans; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D038761:Photobleaching; D011108:Polymers; D021122:Protein Subunits / Q000737:chemistry*
chemical_list: D001896:Boron Compounds; D015220:Calcium Channels; D005456:Fluorescent Dyes; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C522867:Orai3 protein, human; D011108:Polymers; D021122:Protein Subunits; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C109986:2-aminoethoxydiphenyl borate; D016257:Fura-2