We have created a constitutively active, mutant M₃-mACh receptor by single Asn→Tyr point mutation at position 514 ( ^N514YM₃) based on previous work (Ford et al., 2002). When transiently expressed in HEK293 cells, levels of [³H]-inositol phosphate ([³H]-IP_x) accumulation in the presence of 10 mM Li⁺ were approx. three fold greater in ^N514YM₃ compared to wild-type M₃ (M₃-WT) receptor-expressing cells. The mACh receptor antagonists, atropine, 4-DAMP, pirenzepine and methoctramine, were all able to concentration-dependently decrease basal [³H]-IP_x accumulation to levels seen in the absence of Li⁺ in ^N514YM₃ receptor-transfected HEK cells suggesting that these compounds are ‘full’ inverse agonists.

The constitutive activity of the ^N514YM₃ receptor mutant allowed us to investigate a different functional assay to examine further the mechanism(s) of inverse agonism. In preliminary studies it was noted that to achieve equivalent expression levels of the M₃-WT and ^N514YM₃ receptors, significantly greater concentrations of ^N514YM₃ cDNA were required. It has been previously shown that constitutively active receptors are often expressed at lower levels than cognate wild-type receptors, and that expression increases in the presence of an inverse agonist (Smit et al., 1996). Therefore, the expression levels of ^N514YM₃ and M₃-WT receptors were assessed after a 24 h pre-incubation with maximal concentrations of each antagonist (methoctramine was excluded, as it could not readily be washed from cells to allow assessment of changes in receptor number). None of the antagonists tested had a significant effect on the expression level of the M₃-WT receptor; however, the ^N514YM₃ receptor was up-regulated by incubation with atropine (1 µM ) or pirenzepine (30 µM ) (by 93% ± 3 and 84% ± 2, respectively). In contrast, 4-DAMP (1 µM ) caused a more modest up-regulation (47% ± 4).

The possibility that ^N514YM₃ receptor activity may be constitutively down-regulated was assessed by quantifying the relative basal phosphorylation of mutant and wild-type receptors. Basal phosphorylation was significantly greater for the ^N514YM₃ than the M₃-WT receptor (12898 ± 496 and 8304 ± 412 absorbance units, respectively). In addition, atropine, 4-DAMP and pirenzepine were able to decrease significantly the basal phosphorylation of the ^N514YM₃ receptor (by 63 ± 7%, 76 ± 4% and 72 ± 8%, respectively), whereas methoctramine caused a smaller (23 ± 10%) insignificant decrease in the basal level of receptor phosphorylation.

In conclusion, data obtained from [³H]-IP_x accumulation assays suggest that all of the antagonists tested behave as ‘full’ inverse agonists, and that the simplest mechanism of action could be the stabilization of a common inactive (R) receptor conformation. However, data obtained from other assays of inverse agonist efficacy suggest that this may be an over-simplification and that different mechanisms of inverse agonism may exist.

Ford, D.J. et al. (2002) J. Pharmacol. Exp. Ther. , 300, 810-817.
Smit, M.J. et al. (1996) Proc. Natl. Acad. Sci. USA , 93, 6802-6807 .