Allosteric Modulation of Muscarinic Receptor Signaling: Alcuronium-Induced Conversion of Pilocarpine from an Agonist into an Antagonist

Abstract

Previous studies on allosteric interactions at muscarinic receptors have often focused on ligand-receptor binding interactions, because ligand binding seemed to reflect functional consequences. The prototypal allosteric agent alcuronium is known to bind with similar affinity to the M₂ subtype of muscarinic acetylcholine receptors whether or not the receptors are occupied by the agonist pilocarpine. To determine allosteric modulation of receptor signaling by alcuronium, the effects of pilocarpine were measured in contracting guinea pig left atria and on G-protein coupling in M₂-transfected Chinese hamster ovary (CHO) cell membranes. Alcuronium dose-dependently suppressed pilocarpine-induced reduction of isometric contraction force in atria (pIC_50, Alc = 5.63) without any effect on the EC₅₀ of pilocarpine, consistent with an allosteric mechanism. In contrast, alcuronium shifted the concentration-effect curve of the agonist oxotremorine M to the right without affecting the maximal effect, in a formally competitive manner (pK_A, Alc = 5.54). If pilocarpine remained receptor bound in the presence of alcuronium, this indicates that pilocarpine can no longer act as an agonist. In support of this hypothesis, pilocarpine acted as a competitive antagonist against oxotremorine M in the presence of 10 μM alcuronium. Measuring guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in CHO-M₂ membranes yielded similar results. Alcuronium suppressed pilocarpine-induced stimulation of [³⁵S]GTPγS binding (pIC_50, Alc = 5.47) without shift in EC₅₀, whereas it competitively shifted the response to oxotremorine M (pK_A, Alc = 5.97). [³H]Oxotremorine M binding data corresponded with the functional findings. In conclusion, alcuronium converted the agonist pilocarpine into an antagonist—a novel type of functional allosteric interaction.