May Lauren T, Avlani Vimesh A, Langmead Christopher J, Herdon Hugh J, Wood Martyn D, Sexton Patrick M, Christopoulos Arthur
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia.
Mol Pharmacol. 2007 Aug;72(2):463-76. doi: 10.1124/mol.107.037630. Epub 2007 May 24.
The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Tyr(177), and Thr(423). 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 (172)EDGE(175) sequence had been neutralized and each Tyr(177) and Thr(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 (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'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) 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(177) and Thr(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.
M2毒蕈碱型乙酰胆碱受体（mAChR）具有至少一个变构调节剂结合位点，该位点依赖于（172）EDGE（175）、Tyr（177）和Thr（423）残基。然而，与调节剂不同，这些残基对变构激动剂作用的贡献尚不清楚。我们构建了突变型M2 mAChR，其中（172）EDGE（175）序列的电荷被中和，每个Tyr（177）和Thr（423）被丙氨酸取代。放射性配体结合实验表明，这些突变对典型调节剂加拉明、阿库氯铵和庚烷-1,7-双-[二甲基-3'-邻苯二甲酰亚胺基丙基]-溴化铵（C7/3-邻苯二甲酰亚胺）有显著抑制作用，但对正构拮抗剂[3H]N-甲基东莨菪碱影响极小。相比之下，变构激动剂4-I-[3-氯苯基]氨基甲酰氧基)-2-丁炔基三甲基氯化铵（McN-A-343）、4-正丁基-1-[4-(2-甲基苯基)-4-氧代-1-丁基]哌啶盐酸盐（AC-42）以及新型AC-42衍生物1-[3-(4-丁基-1-哌啶基)丙基]-3,4-二氢-2(1H)-喹啉酮（77-LH-28-1）在EDGE-YT联合突变时显示出高亲和力位点的亲和力增加或比例增加，表明其与典型调节剂的结合模式不同。随后对细胞外信号调节激酶（ERK）1/2磷酸化和鸟苷5'-（γ-[(35)S]硫代）三磷酸（[(35)S]GTPγS）结合的功能测定表明，这些突变对正构激动剂乙酰胆碱（ACh）和毛果芸香碱影响极小，但McN-A-343的效力和77-LH-28-1的效能显著增加。额外的诱变实验发现，这些作用主要由Tyr（177）和Thr（423）介导，而非（172）EDGE（175）序列。每种变构激动剂与ACh之间的功能相互作用具有高度负协同性，但与EDGE-YT联合突变型M2 mAChR上变构激动剂亲和力增加一致。因此，本研究揭示了M2 mAChR变构激动剂与变构调节剂结合和功能的关键变构位点残基的不同作用。
