title: State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.
abstract: After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1-Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB-activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd(2+)). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB-activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd(2+)-induced block that is state dependent: Cd(2+) only blocks 2-APB-activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB-activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697.
authors.forename: Anna
authors.initials: A
authors.lastname: Amcheslavsky
authors.forename: Olga
authors.initials: O
authors.lastname: Safrina
authors.forename: Michael D
authors.initials: MD
authors.lastname: Cahalan
mesh_terms: D000595:Amino Acid Sequence; D001896:Boron Compounds / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary
chemical_list: D001896:Boron Compounds; D015220:Calcium Channels; C522867:Orai3 protein, human; C109986:2-aminoethoxydiphenyl borate; D003545:Cysteine
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