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

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha 7 homo-oligomeric and alpha 4 beta 2 heterooligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha 4 subunit were introduced into the alpha 7 agonist binding site, carried by the homo-oligomeric alpha 7-V201-5HT(3) chimera. Introduction of the alpha 4 residues 183-191 into alpha 7 subunit sequence (chimera C-2) 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 alpha 4 residues 151-155 (chimera B) produced a similar to 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 C-2 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 alpha 4 amino acids 151-155 and 183-191 into the alpha 7-V201-5HT(3) chimera thus confers physiological and pharmacological properties typical of the alpha 4 beta 2 receptor.