Critical elements determining diversity in agonist binding and desensitization of neuronal nicotinic acetylcholine receptors

Abstract

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the α7 homo-oligomeric and α4β2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the α4 subunit were introduced into the α7 agonist binding site, carried by the homo-oligomeric α7-V201-5HT3 chimera. Introduction of the α4 residues 183- 191 into α7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the α4 residues 151-155 (chimera B) produced a ~100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to seven fold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of α4 amino acids 151-155 and 183-191 into the α7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the α4/β2 receptor.