Received for publication September 12, 2006.
Revised October 25, 2006.
Accepted for publication October 25, 2006.

Species selectivity of a nicotinic acetylcholine receptor agonist (TMAQ) is conferred by two adjacent extracellular 4 amino acids which are implicated in the coupling of binding to channel gating

Abstract

TMAQ (5-(trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for 4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human 4 subunit but having no detectable agonist activity on nAChRs containing the rat 4 subunit. With the aim of identifying subunit domains and individual amino acids which contribute to the species selectivity of TMAQ, a series of chimeric and mutated 4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric or mutated 4 subunits have been examined by radioligand binding, intracellular calcium assays and electrophysiological recording. Two adjacent amino acids located within the extracellular loop D domain of the 4 subunit (amino acids 55 and 56) have been identified as playing a critical role in determining the agonist potency of TMAQ. Mutagenesis of these two residues within the rat 4 subunit to the corresponding amino acids in the human 4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human 4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the non-selective nicotinic agonist epibatidine. Despite acting as a potent agonist of human 4-containing nAChRs, TMAQ acts as an antagonist of rat 4-containing receptors. Our experimental data, together with homology models of the rat and human 34 nAChRs, suggests that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.

Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants

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