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 alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alpha Y198F and epsilon D175N, by an increase in the equilibrium dissociation constant for ACh (K-D) and a reduction in Theta in alpha Y93F, and only by a reduction in K-D in epsilon E184Q. 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 epsilon E184 showed that K+ competition was unaltered in epsilon E184D and was virtually eliminated in epsilon E184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilon E184 side chain and K+ (similar to 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.