A Mutational Analysis of the Acetylcholine Receptor Channel Transmitter Binding Site

AbstractMutagenesis 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 (KD) and a reduction in Θ in αY93F, and only by a reduction in KD 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+ (∼1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR