Mutation of conserved residues in the M2 domain yields a alpha9 alpha10 nicotinic receptor with a gain of function

PLAZAS, PV; KATZ, E; ELGOYHEN, AB

San Carlos de Bariloche, Argentina

Congreso; Argentine Society for Biochemistry and Molecular Biology XXXIX Annual Meeting, Biophysical Society of Argentina XXXII Annual Meeting, Bariloche Protein Symposium; 2003

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 a9a10 nAChR function. cDNAs for rat a9 and a10 subunits, where 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 a9a10(L9´T) and a9a10(V13´T) nAChRs exhibited much lower desensitization kinetics and an increase in their apparent affinity 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 type receptor, elicited ionic currents in oocytes expressing these mutants. A constitutive activation of a fraction of mutant receptors was observed, even in the absence of the agonist. Our results suggest that the sites where these two residues are located are structurally critical for opening-closing transitions of the a9a10 nAChR.