title: Proton-mediated conformational changes in an acid-sensing ion channel.
abstract: Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC50 by 0.2 pH units, the double mutant D238A/E239A shifted the EC50 by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.
authors.affiliation: From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, and.
authors.forename: Swarna S
authors.initials: SS
authors.lastname: Ramaswamy
authors.forename: David M
authors.initials: DM
authors.lastname: MacLean
authors.forename: Alemayehu A
authors.initials: AA
authors.lastname: Gorfe
authors.forename: Vasanthi
authors.initials: V
authors.lastname: Jayaraman
mesh_terms: D062565:Acid Sensing Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D062565:Acid Sensing Ion Channels; D011522:Protons
keywords: Acid-sensing Ion Channels (ASICs); Fluorescence Resonance Energy Transfer (FRET); Ligand-binding Protein; Ligand-gated Ion Channels; Receptor Activation; Receptor Desensitization; Receptor Structure-Function