Proper determination of agonist efficacy is essential in the assessment of agonist selectivity and signalling bias. Agonist efficacy is a relative term that is dependent on the system in which it is measured, especially being dependent on receptor expression level. The operational model (OM) of functional receptor agonism is a useful means for the determination of agonist functional efficacy using the maximal response to agonist and ratio of agonist functional potency to its equilibrium dissociation constant (KA) at the active state of the receptor. However, the functional efficacy parameter τ is inter-dependent on two other parameters of OM; agonist’s KA and the highest response that could be evoked in the system by any stimulus (EMAX). Thus, fitting of OM to functional response data is a tricky process. In this work we analyse pitfalls of fitting OM to experimental data and propose a rigorous fitting procedure where KA and EMAX are derived from half-efficient concentration of agonist and apparent maximal responses obtained from a series of functional response curves. Subsequently, OM with fixed KA and EMAX is fitted to functional response data to obtain τ. The procedure was verified at M2 and M4 muscarinic receptors fused with the G15 G-protein α-subunit. The procedure, however, is applicable to any receptor-effector system.

The overproduction of β-amyloid (Aβ) fragments in transgenic APPswe/PS1dE9 mice results in formation of amyloid deposits in the cerebral cortex and hippocampus starting around four months of age and leading to cognitive impairment much later. We have previously found an age and transgene-dependent weakening of muscarinic receptor-mediated transmission that was not present in young (6-10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Aβ(1-42) on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 μM Aβ(1-42) had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Aβ(1-42) significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the G(s), G(q/11), and G(i/o) G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Aβ(1-42) and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Aβ(1-42) on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with G(q/11) G-protein.

Methoctramine (N,-methyl]hexyl]-1,8-octane] diamine) is an M 2 -selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M 2 and M 3 receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M 2 receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMSoccupied M 2 receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M 3 receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M 3 receptor.