title: Coupling of proton binding in extracellular domain to channel gating in acid-sensing ion channel.
abstract: Protonation of several amino acid residues in the extracellular domain (ECD) of acid-sensing ion channel (ASIC) causes conformational changes that lead to opening of the channel. It is not clear how conformational changes in ECD are coupled to channel gating. Here, we show that the loop connecting β9 and α4 at the base of the thumb region of ECD interacts with post-TM1 to stabilize the channel in the closed state. Flexibility of these two regions is important for optimum gating of the channel. In ASIC1a, when Y71 (post-TM1) and W287 (β9-α4 loop) were mutated to cysteine, they formed disulfide bond in the closed state. Breaking of the disulfide bond by reducing agent dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) potentiated the current significantly. Engineered cysteine G288C reacted with sulfhydryl-specific methanethiosulfonate ethyltrimethylammonium (MTSET) in the open state but not in closed/steady desensitized state, suggesting gating-associated conformational movement of this loop. We also identified a salt bridge between highly conserved R64 at TM1 and D432 at TM2 that is important for optimum gating. Based on our results and other published work, we propose that proton binding in ECD is followed by the displacement of the β9-α4 loop of the thumb, leading to the rotation of TM1. Conformational movement propagates to TM2 and the channel gate opens by the concomitant movement of TM2 and breaking of the salt bridge between R64 and D432.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, Tamil Nadu, India.
authors.forename: Sandip Madhusudan
authors.initials: SM
authors.lastname: Swain
authors.forename: Amal Kanti
authors.initials: AK
authors.lastname: Bera
mesh_terms: D062565:Acid Sensing Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D051381:Rats
chemical_list: D062565:Acid Sensing Ion Channels; C568268:Asic1 protein, rat; D011522:Protons; D003545:Cysteine