PROTON MAGNETIC-RESONANCE STUDIES OF CHOLINERGIC LIGAND-BINDING TO THE ACETYLCHOLINE-RECEPTOR IN ITS MEMBRANE ENVIRONMENT

Proton magnetic resonance has been used to monitor binding of choline, a known partial agonist, to acetylcholine receptor-enriched membrane preparations from Torpedo californica electroplax. The interaction between choline and receptor led to a broadening of the resonance of the choline methyl groups and this effect was reversed by α-bungarotoxin, a quasi-irreversible antagonist of the acetylcholine receptor. From the concentration dependence of line broadening the equilibrium dissociation constant for choline was obtained (K(d) = 190 ± 65 μM). The temperature dependence of the parameters observed in the choline titrations gave an enthalpy of binding ΔH < 1.5 kcal/mol and allowed estimates for the dissociation rate constant of the receptor-choline complex (k(diss > 1.6 x 103 S1) and the respective activation energy, E(a) (k(diss ~ 5.5 kcal/mol. The association of other ligands with the membrane-bound receptor could also be studied by observing effects of varying concentrations of such ligands on the choline methyl group linewidth at a constant choline concentration.