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

• Published in: Molecular pharmacology Vol. 70; no. 1; p. 181
• Main Authors: Prilla, Stefanie, Schrobang, Jasmin, Ellis, John, Höltje, Hans-Dieter, Mohr, Klaus
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.07.2006
• Subjects: Allosteric Regulation - drug effects; Allosteric Site; Amino Acid Sequence; Binding Sites - genetics; Binding, Competitive; Gallamine Triethiodide - chemistry; Gallamine Triethiodide - metabolism; Gallamine Triethiodide - pharmacology; Humans; Isoindoles; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation - genetics; N-Methylscopolamine - chemistry; N-Methylscopolamine - metabolism; N-Methylscopolamine - pharmacology; Phthalimides - chemistry; Phthalimides - metabolism; Phthalimides - pharmacology; Receptors, Muscarinic - chemistry; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Threonine - genetics; Threonine - metabolism; Tryptophan - genetics; Tryptophan - metabolism
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.106.023481

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.