Abstract

The M₂ muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues ¹⁷²EDGE¹⁷⁵, Tyr¹⁷⁷, and Thr⁴²³. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M₂ mAChRs in which the charge of the ¹⁷²EDGE¹⁷⁵ sequence had been neutralized and each Tyr¹⁷⁷ and Thr⁴²³ was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C₇/3-phth) but minimal effects on the orthosteric antagonist [³H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (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 extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (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 Tyr¹⁷⁷ and Thr⁴²³, rather than the ¹⁷²EDGE¹⁷⁵ 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.