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CELLULAR AND MOLECULAR

Constitutive Activity and Inverse Agonism at the M₂ Muscarinic Acetylcholine Receptor

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M₂ muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [³H]inositol phosphates in cells coexpressing the chimeric G_qi5 protein and the N410Y mutant M₂ mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M₂ mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M₂ mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [³H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of G_i/o versus G_s, consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M₂ mACh receptor. This indicates that the N410Y mutation of the M₂ mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.

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

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