Mutation of conserved residues in the M2 domain alter channel gating of the alpha9alpha10 nicotinic receptor

ELGOYHEN AB

Angra dos Reis, Brasil

Congreso; 1st Latin American Protein Society Meeting, Symposium: The Intimacies of Ion Channels: Structure and Function; 2004

Latin American Protein Society

Nicotinic acetylcholine receptors (nAChRs) form part of a gene superfamily, which includes GABAA, GABAc, serotonin type 3 and glycine receptors. The putative channel-forming M2 domains of these receptors contain two highly conserved residues: a leucine (L9) and a valine (V13), which are postulated to form a constricting hydrophobic girdle in the middle of the ion pathway. The aim of the present work was to study the role of these residues in the alpha9alpha10 nAChR function. cDNAs from rat alpha9 and alpha10 subunits, in which the amino acids L9 or V13 were mutated to threonine (T), were expressed in Xenopus laevis oocytes and agonist-evoked currents were measured under two-electrode voltage-clamp. When compared to wild type receptors, ACh-evoked currents through alpha9alpha10(L9T) and alpha9alpha10(V13T) nAChRs exhibited much lower desensitization kinetics and an increase in their apparent anity for this agonist. Choline, a weak partial agonist of the wild type receptor, behaved as a full agonist of the mutant receptors. Furthermore, nicotine, muscarine and ICS-205 930, antagonists of the wild typereceptor, elicited ionic currents in oocytes expressing these mutants. A constitutive activation of mutant receptors was observed, even in the absence of the agonist. Single channel recordings in the cell-attached mode in oocytes, revealed an increase in the tau-on of the mutants, being more pronounced in the case of V13 and indicating a stabilization of the open state. Our results suggest that sites centrally located at the TM2 region of the pore are involved in channelgating of the alpha9alpha10 nAChR.