A mutational analysis of the acetylcholine receptor channel transmitter binding site

Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC₅₀ of the ACh dose-response curve was increased following α-subunit mutations Y93F and Y198F and ε-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Θ) in αY198F and εD175N, by an increase in the equilibrium dissociation constant for ACh (K(D)) and a reduction in Θ in αY93F, and only by a reduction in K(D) in εE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K⁺. Additional mutations of εE184 showed that K⁺ competition was unaltered in εE184D and was virtually eliminated in εE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the εE184 side chain and K⁺ (~ 1.7k(B)T), but not ACh⁺. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR.