Agonist-mediated conformational changes of muscarinic receptors in rat brain

Muscarinic acetylcholine receptors were identified in the microsomal P fraction of rat forebrain by the specific binding of the radiolabeled antagonist [3H]dexetimide. Binding occurred to a single class of noncooperative sites (3.25 mumol/mg protein) with an equilibrium dissociation constant of 1.1 nM. Agonist/[3H]-dexetimide competition binding experiments allowed the distinction between two major muscarinic receptor subpopulations, having respectively high affinity (20% of the total receptor population) and low affinity for agonists, but with the same affinity for antagonists. A 610-fold difference in affinity was calculated for carbamoyl-choline, the agonist extensively investigated in this study. The alkylating reagent N-ethylmaleimide did not affect the total receptor number, antagonist binding to the high-affinity and low-affinity sites, nor agonist binding to the high-affinity sites. The reagent, however, caused a net increase in agonist affinity for the low-affinity sites. This process was dependent on time and dose of N-ethylmaleimide, until a maximal increase in affinity (fourfold increase for carbamoylcholine) was attained. This suggests a quantal conversion of the low-affinity sites by the reagent into an alkylated form, which possesses a higher affinity for agonists but an unchanged affinity for antagonists. The rate of alkylation was enhanced by the presence of agonists but not of antagonists, which is indicative for the ability of agonists to mediate a conformational change of these sites. The close correlation between the N-ethylmaleimide-mediated increase in drug affinity for the low-affinity sites and the ability of the drugs to enhance alkylation of these sites by N-ethylmaleimide can be explained by the ability of (a) muscarinic drugs to interact with the low-affinity sites according to the Monod-Wyman-Changeux 'Plausible Model' and (b) N-ethylmaleimide to freeze these sites in the 'active' conformation by alkylation.