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 (EC₅₀) and efficacy (maximum response, I_max) are well-studied, the role of unliganded gating (minimum response, I_min) 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_min in modulating EC₅₀ and I_max. Three Gain-of-Function (GOF) mutations that increase, and two Loss-of-Function (LOF) mutations that decrease the unliganded gating equilibrium constant (L₀) 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 L₀. 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 (L₀) 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 L₀. The study highlights the importance of intrinsic activity in receptor theory.