CONICET | Buscador de Institutos y Recursos Humanos

Nicotinic acetylcholine receptors are a family of ligand-gated non-selective cationic channels that participate in fundamental physiological processes both at the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The á9á10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family since it shows differences in the extent of calcium permeability across species. In particular, mammalian á9á10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This evolutionarily-acquired adaptation provides the unique opportunity of studying how protein function was shaped along evolutionary history, by identifying amino acids that were positively selected by nature. 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 evolution events that yielded a high calcium permeable nicotinic á9á10 mammalian receptor. Only three specific amino acid substitutions in the á9 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 were selected amongst other seemingly neutral changes that provided the background structure necessary for high calcium permeability. Thus, the consequences of a given substitution greatly depend on the overall sequence (and hence structure) of the protein, a phenomena referred to as epistasis These results highlight the importance of tracking evolutionarily-acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.