This paper was published as Journal of Pharmacology and Experimental Therapeutics, 2006, 316 (1), pp. 279-288. It is available from http://jpet.aspetjournals.org/content/316/1/279. Doi: 10.1124/jpet.105.094383Metadata only entryIntroduction 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 Gαqi5 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 Gαi/o versus Gαs, 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
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