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-d irected mutagenesis of these residues (alpha Y93, alpha Y190, and alph a Y198; the notation used is subunit/amino acid/position in the Torped o receptor/substitution) in mouse muscle, Torpedo electroplax, and alp ha 7 neuronal acetylcholine receptors have demonstrated that substitut ion of phenylalanine for tyrosine results in a shift towards higher co ncentrations in the macroscopic dose-response curves for acetylcholine -elicited currents from voltage-clamped Xenopus oocytes that express t he 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 analy sis of ion channel gating at the single-channel level to obtain estima tes for the rate constants associated with the ligand binding and chan nel opening steps for wild-type, alpha Y93F, and alpha Y198F receptors . The results suggest that the underlying cause of the shifts in the m acroscopic dose-response curves is a reduction in acetylcholine affini ty for the resting activatable state of the receptor. Furthermore, it is the association rate for agonist binding, rather than the dissociat ion rate, that is most affected by the mutations.