Received for publication February 13, 2006.
Revised April 18, 2006.
Accepted for publication April 26, 2006.

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

Abstract

Generally, 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 M₂¹⁷⁷Tyr and M₂⁴²³Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M₂⁴²²Trp and the adjacent M₂⁴²³Thr 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 M₂⁴²²Trp and M₂⁴²³Thr independently influenced allosteric agent binding. The presence of M₂⁴²³Thr 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 M₂⁴²²Trp and of the corresponding M₅⁴⁷⁷Trp 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 M₂¹⁷⁷Tyr and M₂⁴²²Trp, in a concerted action, may fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M₂⁴²²Trp and the spatially adjacent M₂¹⁷⁷Tyr, as well as M₂⁴²³Thr, 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.

Key words: Muscarinic cholinergic, Mutagenesis/Chimeric approaches, Receptor binding studies

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