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CELLULAR AND MOLECULAR

A Single Point Mutation (N514Y) in the Human M₃ Muscarinic Acetylcholine Receptor Reveals Differences in the Properties of Antagonists: Evidence for Differential Inverse Agonism

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (M.R.D., J.M.W., D.C.B., S.R.N., R.A.J.C.); and Novartis Horsham Research Centre, Horsham, West Sussex, United Kingdom (M.R.D., D.C.B., S.J.C.)

A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (300%) in agonist-independent [³H]inositol phosphate ([³H]IP_x) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M₃ and ^N514YM₃ mACh receptor-mediated basal [³H]IP_x accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the ^N514YM₃ mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate ^N514YM₃ mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the ^N514YM₃ mACh receptor was approximately 100% greater than that seen for the WT-M₃ receptor. The ability of antagonists to decrease basal ^N514YM₃ mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [³H]IP_x accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

Address correspondence to: R. A. J. Challiss, Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester, University Road, Leicester, LE1 9HN, UK. E-mail: jc36{at}leicester.ac.uk