A mutational analysis of the acetylcholine receptor channel transmitter binding site

Mutagenesis and single-channel kinetic analysis were used to investigate th e roles of four acetylcholine receptor channel (AChR) residues that are can didates for interacting directly with the agonist. The EC50 of the ACh dose -response curve was increased following alpha-subunit mutations Y93F and Y1 98F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic m odeling indicates that the increase was caused mainly by a reduced gating e quilibrium constant (Theta) in alpha Y198F and epsilon D175N, by an increas e 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 epsi lon E184D and was virtually eliminated in epsilon E184K, but that neither o f 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 term s of multisite and induced-fit models of ligand binding to the AChR.