Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors

After short preincubations with N-[H-3]methylscopolamine ([H-3]NMS) or R(-) -[H-3]quinuclidinyl benzilate ([H-3]QNB), radioligand dissociation from mus carinic M-1 receptors in Chinese hamster ovary cell membranes was fast, mon oexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation w as slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand, Apparently, the unlabeled ligand becomes able to ass ociate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dis sociation. When the membranes were preincubated with [H-3]NMS and then expo sed to benzilylcholine mustard (covalently binding specific ligand), [H-3]N MS dissociation was blocked in wild-type receptors, but not in mutated (D99 N) M-1 receptors, Covalently binding [H-3]propylbenzilylcholine mustard det ected substantially more binding sites than [H-3]NMS. The observations supp ort a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and tra nslocate to the central subsite (binding with higher affinity). The periphe ral subsite of M-1 receptors may include Asp-99, Experimental data on [H-3] NMS and [H-3]QNB association and dissociation perfectly agree with the pred ictions of the tandem two site model.