Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Y(177) and T(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs where the charge of the (172)EDGE(175) sequence had been neutralized and each of Y(177) and T(423) were substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium and C7/3-phth, but minimal effects on the orthosteric antagonist, [(3)H]NMS. In contrast, the allosteric agonists, McN-A-343, AC-42 and a novel AC-42 derivative, 77-LH-28-1, demonstrated an increased affinity or proportion of high affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of ERK1/2 phosphorylation and [(35)S]GTPgammaS binding revealed minimal effects of the mutations on the orthosteric agonists, ACh and pilocarpine, but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Y(177) and T(423), rather than the (172)EDGE(175) sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity, but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.