Goodwin, J.A.; (2006) Molecular basis of ligand binding to the M1 muscarinic acetylcholine receptor: Focus on the second extracellular loop. Doctoral thesis , University of London.

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Abstract

The second extracellular loop (E2) of the Mi muscarinic acetylcholine receptor (mAChR) links transmembrane (TM) helices 4 and 5 and is predicted to play a role in the acetylcholine (ACh) binding pocket. Potential ligand contact residues were selected using a rhodopsin homology model of the Mi mAChR and mutated to alanine. Cys98, postulated to form a disulphide (S-S) anchor to Cysl78, Asp99, possibly part of a ligand access channel, and Phel97 and Trp378, predicted to complete the floor of the binding pocket, were also mutated. The double cysteine mutant was also generated. Radioligand binding assays showed that COS-7 cells expressed mutants at near to wild-type (WT) levels except for Cys 98 Ala, Cysl78Ala and Asp99Ala (10-20% of WT). The affinity of the antagonist (-)-N-methylscopolamine (NMS) was reduced by Cys98Ala (40-fold), Asp99Ala (22-fold), and Cysl78Ala (16 fold). ACh affmity was reduced by Asp99Ala (11-fold), Cys98Ala (12-fold), Argl71Ala (7-fold), Cysl78Ala (70-fold) and Ilel80Ala (5-fold). The TM domain mutants Phel97Ala and Trp378Ala reduced NMS affinity 90 and 400-fold respectively but reduced ACh affinity only 2-fold and 13-fold. The mutants, supplemented with six E2 loop mutants from a previous study were able to activate the Gq-linked phosphoinositide signalling pathway. Cysteine mutations produced greater than 2500-fold reductions in ACh potency resulting in a 10-20-fold loss of signalling efficacy. Effects were also observed for other E2 loop mutants, most notably a 9-fold loss of efficacy for Serl84Ala. Dissociation rate constants for NMS and 3-Quinuclidinyl Benzilate (QNB) were increased by the majority of mutations, Argl71Ala, Cysl78Ala and Trp378Ala being the most significant. The results imply that the E2 loop plays a more important role in the binding of ACh than NMS while NMS may bind deeper within the TM domain than ACh. Maintenance of the S-S bond was essential for receptor activation. Studies with additional ligands showed that pilocarpine, oxotremorine-M and oxotremorine followed an ACh-like binding pattern, whereas the atypical agonists McN-A-343 and AC-42 may bind outside the ACh binding pocket.

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