title: A mutational analysis of the acetylcholine receptor channel transmitter binding site.
abstract: 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 alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. 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 epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR.
authors.affiliation: Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214 USA.
authors.forename: G
authors.initials: G
authors.lastname: Akk
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: A
authors.initials: A
authors.lastname: Auerbach
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008564:Membrane Potentials; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection
chemical_list: D007473:Ion Channels; D008024:Ligands; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D012964:Sodium; D000109:Acetylcholine; D011188:Potassium