Abstract

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M₁ receptor previously suggested to interact with an “ectopic” site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M₁ receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [³H]N-methylscopolamine ([³H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [³H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [³H]NMS dissociation from CHO-hM₁ cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M₁ receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M₁ receptors in the absence of the orthosteric agonist.