The effect of unliganded gating on agonist response in nicotinic receptors.

Understanding the agonist concentration-response curve (CRC) is the cornerstone in pharmacology. While CRC parameters, agonist potency (EC) and efficacy (maximum response, I) are well-studied, the role of unliganded gating (minimum response, I) on CRC is often overlooked. This study explores the effect of unliganded gating on agonist response in muscle-type acetylcholine (ACh) receptors, focusing on the underexplored role of I in modulating EC and I. Three Gain-of-Function (GOF) mutations that increase, and two Loss-of-Function (LOF) mutations that decrease the unliganded gating equilibrium constant (L) were studied using automated patch-clamp electrophysiology. GOF mutations enhanced agonist potency, whereas LOF mutations reduced it. The calculated CRC aligned well with empirical results, indicating that agonist CRC can be estimated from knowledge of L. Reduction in agonist efficacy due to LOF mutations was calculated and subsequently validated using single-channel patch-clamp electrophysiology, a factor often obscured in normalized CRC. The study also evaluated the combined impact of mutations (L) on CRC, confirming the predictive model. Further, no significant energetic coupling between distant residues (>15 Å) was found, indicating that the mutations' effects are localized and do not alter overall agonist affinity. These findings substantiate the role of unliganded gating in modulating agonist responses and establishes a predictive model for estimating CRC parameters from known changes in L. The study highlights the importance of intrinsic activity in receptor theory.