STABLE ALLOSTERIC BINDING OF M1-TOXIN TO M1 MUSCARINIC RECEPTORS

m1-Toxin was found to slow the dissociation of [H-3]N-methylscopolamin e (NMS) and [H-3]pirenzepine from m1 muscarinic receptors expressed in the membranes of Chinese hamster ovary cells. When toxin-NMS-receptor complexes were formed in membranes and then dissolved in digitonin, o r when these complexes were formed in solution, the toxin completely s topped the dissociation of [H-3]NMS for 6 hr at 25-degrees-C. Toxin-re ceptor complexes formed in membranes or in solution were also highly s table in solution at 25-degrees, as shown by the ability of the toxin to prevent the binding of [H-3]quinuclidinyl benzilate (QNB). [H-3] QN B-receptor complexes were equally stable, whereas unliganded soluble r eceptors lost most of their ability to bind QNB within an hour. Toxin- receptor complexes could be partially dissociated by incubation at 37- degrees in the presence of digitonin and [H-3]QNB, and the freed recep tors were then labeled. These results demonstrate that m1-toxin binds allosterically and pseudoirreversibly to m1 receptors, and that the to xin can stabilize the outward-facing pocket of m1 receptors which cont ains and binds competitive antagonists. The allosteric nature of the b inding of m1-toxin should prove to be useful for such unusual purposes as stabilizing the binding of readily reversible and/or nonselective ligands specifically to m1 receptors, for purifying labeled or unlabel ed receptors by affinity techniques which recognize the toxin, for rec ognizing receptors with genetically or biochemically altered primary b inding sites, and for stabilization of the native conformation of m1 r eceptors for structural studies.