title: Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.
abstract: Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Dr. Héctor N Torres, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina marcela.lipovsek@gmail.com.
authors.forename: Marcela
authors.initials: M
authors.lastname: Lipovsek
authors.affiliation: Department of Organic Chemistry, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: Angélica
authors.initials: A
authors.lastname: Fierro
authors.affiliation: Department of Organic Chemistry, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: Edwin G
authors.initials: EG
authors.lastname: Pérez
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Dr. Héctor N Torres, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Boffi
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom.
authors.forename: Neil S
authors.initials: NS
authors.lastname: Millar
authors.affiliation: Department of Otolaryngology, Head and Neck Surgery, and Center for Hearing and Balance, The Johns Hopkins University School of Medicine, Baltimore, MD.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fuchs
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Dr. Héctor N Torres, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Katz
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Dr. Héctor N Torres, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina Departamento de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Ana Belén
authors.initials: AB
authors.lastname: Elgoyhen
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D019143:Evolution, Molecular; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000494:pharmacology; D010539:Permeability; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D030161:Avian Proteins; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D000109:Acetylcholine; D002118:Calcium
keywords: calcium permeability; hearing; molecular evolution; nicotinic receptors