Abstract：
Understanding the agonist concentration-response curve (CRC) is the cornerstone in pharmacology. While CRC parameters, agonist potency (EC50) and efficacy (maximum response, Imax) are well-studied, the role of unliganded gating (minimum response, Imin) 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 Imin in modulating EC50 and Imax. 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.
摘要：
了解激动剂浓度-反应曲线（CRC）是药理学的基石。虽然CRC参数，激动剂效力(EC50)和功效(最大反应，Imax)得到了充分的研究，无配体门控的作用(最小响应，Imin）在CRC上经常被忽视。这项研究探讨了无配体门控对肌肉型乙酰胆碱（ACh）受体激动剂反应的影响，重点研究Imin在调节EC50和Imax中的作用。三个增加的功能增益（GOF）突变，使用自动膜片钳电生理学研究了两个降低无配体门控平衡常数（L0）的功能丧失（LOF）突变。GOF突变增强激动剂效力，而LOF突变减少了它。计算的CRC与经验结果吻合良好，表明激动剂CRC可以从L0的知识来估计。计算了由于LOF突变导致的激动剂功效降低，随后使用单通道膜片钳电生理学进行了验证，在标准化CRC中经常被掩盖的因素。该研究还评估了突变（L0）对CRC的综合影响，确认预测模型。Further,在远处的残基之间没有发现明显的能量耦合（>15埃达），表明突变效应是局部的，不会改变总体激动剂亲和力。这些发现证实了无配体门控在调节激动剂应答中的作用,并建立了用于从L0的已知变化估计CRC参数的预测模型。该研究强调了内在活性在受体理论中的重要性。
