Abstract

5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one (TMAQ) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for β4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human β4 subunit but having no detectable agonist activity on nAChRs containing the rat β4 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 β4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric, or mutated β4 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 β4 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 β4 subunit to the corresponding amino acids in the human β4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human β4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the nonselective nicotinic agonist epibatidine. Despite acting as a potent agonist of human β4-containing nAChRs, TMAQ acts as an antagonist of rat β4-containing receptors. Our experimental data, together with homology models of the rat and human α3β4 nAChRs, suggest that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.