Structure-Function Studies of Allosteric Agonism at M sub(2) Muscarinic Acetylcholine Receptors

• Published in: Molecular pharmacology Vol. 72; no. 2; pp. 463 - 476
• Main Authors: May, Lauren T, Avlani, Vimesh A, Langmead, Christopher J, Herdon, Hugh J, Wood, Martyn D, Sexton, Patrick M, Christopoulos, Arthur
• Format: Journal Article
• Language: English
• Published: 01.08.2007
• ISSN: 0026-895X

The M sub(2) muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues super(172)EDGE super(175), Tyr super(177), and Thr super(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M sub(2) mAChRs in which the charge of the super(172)EDGE super(175) sequence had been neutralized and each Tyr super(177) and Thr super(423) 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 (C sub(7)/3-phth) but minimal effects on the orthosteric antagonist [ super(3)H]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'-( gamma -[ super(35)S]thio)triphosphate ([ super(35)S]GTP gamma S) 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 Tyr super(177) and Thr super(423), rather than the super(172)EDGE super(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 M sub(2) 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 M sub(2) mAChRs.