Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2 177Tyr and M2 423Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2 422Trp and the adjacent M2 423Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2 422Trp and M2 423Thr independently influenced allosteric agent binding. The presence of M2 423Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart 478His). Replacement of M2 422Trp and of the corresponding M5 477Trp 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 M2 receptor model revealed that the aromatic rings of M2 177Tyr and M2 422Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2 422Trp and the spatially adjacent M2 177Tyr, as well as M2 423Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 subtype selectivity and baseline affinity of allosteric agents.