Allosteric interactions with muscarinic acetylcholine receptors:: Complex role of the conserved tryptophan M2 422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity

In general, the M-2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M-5 subtype the lowest. The M-2/M-5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M-2 (177)Tyr and M-2 (423)Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M-2 (422)Trp and the adjacent M-2 (423)Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M-2 or M-5 receptors or mutants thereof. It was found that M-2 (422)Trp and M-2 (423)Thr independently influenced allosteric agent binding. The presence of M-2 (423)Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M-5 counterpart (478)His). Replacement of M-2 (422)Trp and of the corresponding M-5 (477)Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMS-bound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M-2 receptor model revealed that the aromatic rings of M-2 (177)Tyr and M-2 (422)Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M-2 (422)Trp and the spatially adjacent M-2 (177)Tyr, as well as M-2 (423)Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M-2/M-5 subtype selectivity and baseline affinity of allosteric agents.