Page, Karen M; (1995) The molecular basis of agonist action at muscarinic acetylcholine receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Muscarinic acetylcholine receptors (mAChRs) are members of the superfamily of G-protein-coupled, seven transmembrane domain, receptors. The aim of this project was to identify amino acids important for the binding of acetylcholine (ACh) to the ground state and activated state of the receptors. A conserved aspartate (Asp105 m1, Asp103 m2) in the third transmembrane domain (TMD III) was mutated to glutamate in the ml and m2 subtypes. Experiments were also performed on an m1 Asp105Asn mutant receptor. The mutations always strongly disfavoured the high affinity, putative ternary, component of agonist binding and the m1 phosphoinositide response. However, their effects on low affinity binding were variable. The binding of ACh itself was strongly inhibited. In contrast, the binding of other ligands was less sensitive, particularly to the Glu mutation. Such ligands were unable to activate the Glu receptor. They may be able to utilise non-productive binding modes, in which their interactions with the binding site are different from those made by ACh. These results suggest that the headgroup of ACh forms an ionic interaction with the Asp in TMD III in the activated state of the receptor. The results also pointed to the importance of the acetyl methyl group of ACh in binding to, and activation of, both the wild-type and Glu mutant receptors. Residues in TMD V, approximately 10-12 Å from the TMD III Asp, were mutated to cysteines in the m1 mAChR, and the receptors probed with BrACh. BrACh has the potential for binding irreversibly to accessible S- groups of cysteines. Irreversible binding was detected at the Thr192Cys mutant receptor, suggesting that Thr192 may contribute to the binding of the terminal methyl group of ACh. The bridging of TMDs III and V may be important in generating the conformational change required for activation.

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