Gating properties of mutant acetylcholine receptors.

A number of affinity labeling studies have identified several tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor as being in or near the ligand binding site. Studies employing site-directed mutagenesis of these residues (alpha Y93, alpha Y190, and alpha Y198; the notation used is subunit/amino acid/position in the Torpedo receptor/substitution) in mouse muscle, Torpedo electroplax, and alpha 7 neuronal acetylcholine receptors have demonstrated that substitution of phenylalanine for tyrosine results in a shift towards higher concentrations in the macroscopic dose-response curves for acetylcholine-elicited currents from voltage-clamped Xenopus oocytes that express the receptors. This decrease in apparent affinity has been ascribed to either a reduction in binding affinity or a reduction in the coupling of agonist binding to ion channel opening; both mechanisms would give rise to shifts in the dose-response curves. We have used kinetic analysis of ion channel gating at the single-channel level to obtain estimates for the rate constants associated with the ligand binding and channel opening steps for wild-type, alpha Y93F, and alpha Y198F receptors. The results suggest that the underlying cause of the shifts in the macroscopic dose-response curves is a reduction in acetylcholine affinity for the resting activatable state of the receptor. Furthermore, it is the association rate for agonist binding, rather than the dissociation rate, that is most affected by the mutations.