Asymmetric Agonist Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A Key Determinant of Agonist Efficacy

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and converging evidence indicates that this type is a key mediator of the rewarding and reinforcing effects of nicotine. nAChR α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)2α4 and (α4β2)2β2 receptors that are activated by agonists with strikingly differing efficacies. Although the alternate receptors contain identical agonist sites at α4/β2 subunit interfaces, they possess isoform-specific subunit interfaces that are thought to underlie receptor stoichiometry functional signatures. Recent breakthroughs have identified an additional functionally relevant agonist binding site at the α4(+)/α4(-) interface that is responsible for the signature sensitivity of (α4β2)2α4 nAChRs to activation by full and partial agonists, yet the structural basis for agonist efficacy at this receptor type is not yet understood. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)2α4 receptor. We show that (a) the agonist site at the α4/α4 interface, but not the sites at α4/β2 interfaces, excludes large agonists; b) agonist effects on individual agonist sites differ depending on the interface location of the agonist sites. We conclude that agonist efficacy at (α4β2)2α4 receptors is first determined by the ability of agonists to bind all agonist sites and then by agonist-specific interactions with the agonist sites. The findings add another level of complexity to the structural mechanisms that govern partial agonism in hetero-pentameric ligand gated ion channels.