title: The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.
abstract: The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Biology and Medicine, Lausanne University, Lausanne, Switzerland.
authors.forename: Miguel Xavier
authors.initials: MX
authors.lastname: van Bemmelen
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Biology and Medicine, Lausanne University, Lausanne, Switzerland.
authors.forename: Delphine
authors.initials: D
authors.lastname: Huser
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Biology and Medicine, Lausanne University, Lausanne, Switzerland.
authors.forename: Ivan
authors.initials: I
authors.lastname: Gautschi
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Biology and Medicine, Lausanne University, Lausanne, Switzerland.
authors.forename: Laurent
authors.initials: L
authors.lastname: Schild
mesh_terms: D062565:Acid Sensing Ion Channels / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000166:cytology; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D013776:Tetrathionic Acid / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: C568266:ASIC1 protein, human; D062565:Acid Sensing Ion Channels; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D013776:Tetrathionic Acid; D003545:Cysteine