Investigating the structural determinants underlying allosteric agonism at M2 muscarinic acetylcholine receptors

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 in transmembrane (TM) domains 3 (W99A, L100A, S107A) and 6 (Y403A), or allosteric‐site mutations in extracellular loop 2/top of TM 7 (Y177A/T423A), and investigated their impact on the function of the putative allosteric agonists McN‐A‐343 and N‐desmethylclozapine (NDMC). As expected, orthosteric site mutations reduced the potency and/or efficacy of the orthosteric agonists ACh and pilocarpine, whereas the allosteric site mutations had no significant effects. In contrast, W99A, Y403A and Y177A/T423A caused significant enhancements in McN‐A‐343 efficacy. A similar profile was noted for NDMC, whereas minimal effects were observed on either allosteric agonist at the L100A and S107A mutants. These findings indicate it is possible to engender active conformations of the M2 mAChR by utilizing epitopes at sites distinct from the orthosteric site. This work is supported by National Health & Medical Research Council Project Grant No. 400134 and the Dowd Foundation.