Ward, Stuart D. C.; (1999) The role of residues Tyr381 to Val387, in transmembrane domain six of the rat M1 muscarinic acetylcholine receptor, in agonist binding and receptor activation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this project was to investigate the role of residues Tyr381 to Val387 in transmembrane domain six of the rat M1 muscarinic acetylcholine receptor. Scanning mutagenesis was used. The mutant receptors were characterised by radioligand binding studies and phosphoinositide turnover assays giving measurements of both acetylcholine binding and the acetylcholine induced functional response. Tyr381Ala and Asn382Ala gave the greatest effects. Interestingly, Tyr381 and Asn382 are both conserved in, and specific to the muscarinic acetylcholine receptors and are, therefore, likely to have important functions. The role of these two residues was investigated further by (i) using three series of agonist and antagonist analogues (ACh analogues, azanorbornane- and quinuclidine-based ligands and atropine analogues) as pharmacological probes and, (ii) making the Tyr381Phe mutation to analyse the function of the hydroxyl group and benzene ring of Tyr381. The results indicated that Tyr381 was important in forming interactions with agonists and subsequent receptor activation, whereas Asn382 seemed to be more important for antagonist, especially atropine analogue, binding. Additionally, both residues may form intra-molecular interactions which help to stabilise receptor folding. In the receptor's ground state, the hydroxyl group of Tyr381 forms a hydrogen-bond interaction with the ester-moiety present in the side-chain of acetylcholine, whereas, the benzene ring of Tyr381 is critical for stabilisation or formation of the receptor's activated state, probably by forming a cation-? interaction with the positively charged head-group. The Tyr381 residue made similar interactions with the azanorbornane- and quinuclidine-based ligands, although with a different balance between ground and activated states. In the case of antagonists related to atropine, Tyr381 made both head-group and side-chain interactions in the receptor's ground state. However, Asn382 seemed to play a bigger role, than Tyr381, in the binding of these compounds, via the formation of a hydrogen-bond interaction with their ester moiety.

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