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 (K_A) at the active state of the receptor. However, the functional efficacy parameter τ is inter-dependent on two other parameters of OM; agonist’s K_A and the highest response that could be evoked in the system by any stimulus (E_MAX). 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 K_A and E_MAX are derived from the half-efficient concentration of agonist and apparent maximal responses obtained from a series of functional response curves. Subsequently, OM with fixed K_A and E_MAX 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.

https://www.nature.com/articles/s41598-019-40993-w