Binding and functional characterisation of allosteric agonists at M2 muscarinic acetylcholine receptors

Abstract

Muscarinic acetylcholine receptors (mAChRs) contain at least one allosteric site that is topographically distinct from the acetylcholine (ACh)‐binding orthosteric site. Although numerous studies have investigated the structural basis of allosteric modulation at these receptors, far less is known about allosteric ligands that activate the receptor in their own right. We generated a number of M2 mAChRs harboring different orthosteric‐site mutations (W99A, Y403A), or allosteric‐site mutations (172EDGE175‐QNGQ, Y177A), and investigated their impact on the binding and function of the putative allosteric agonists AC‐42, 77‐LH‐28‐1, McN‐A‐343 and N‐desmethylclozapine (NDMC). As expected, orthosteric site mutations reduced the affinity and/or efficacy of the orthosteric agonists ACh and pilocarpine, whereas the allosteric site mutations had no significant effects. W99A and Y403A caused significant increases in the affinity (100–300 fold and 3–10 fold respectively) and efficacy of AC‐42 and 77‐LH‐28‐1. NDMC and McN‐A‐343 showed increased efficacy, but not affinity, at Y403A and Y177A. Homology models of the M2 mAChR have been developed around the different ligands. The experimental data and modelling together lead to a better understanding of the allosteric binding sites of mAChRs. This work is supported by National Health & Medical Research Council Project Grant No. 400134 and the Dowd Foundation.