title: Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation.
abstract: We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.
authors.affiliation: Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.
authors.forename: H L
authors.initials: HL
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Auerbach
authors.forename: N
authors.initials: N
authors.lastname: Bren
authors.forename: K
authors.initials: K
authors.lastname: Ohno
authors.forename: A G
authors.initials: AG
authors.lastname: Engel
authors.forename: S M
authors.initials: SM
authors.lastname: Sine
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D051379:Mice; D009154:Mutation / Q000235:genetics*; D018408:Patch-Clamp Techniques; D011950:Receptors, Cholinergic / Q000187:drug effects* / Q000235:genetics*
chemical_list: D011950:Receptors, Cholinergic; D000109:Acetylcholine