The kinetics of competitive antagonism of nicotinic acetylcholine receptors.

摘要

Competitive antagonists of nicotinic acetylcholine receptors (nAChR) are clinically used as muscle relaxants. Very little is known about their kinetics of inhibition. We developed novel rapid perfusion devices to make electrophysiological measurements of kinetics (in absence of ACh) on the millisecond timescale. We determined the binding affinity, and association and dissociation rate constants of (+)-tubocurarine, pancuronium, rapacuronium, metocurine, cisatracurium and gallamine. Association and dissociation rate constants (25°C) ranged from 1.6 x 109M-1s -1 to 7.5 x 107M-1s -1 and 2s-1 to 1700s-1, respectively, consistent with binding affinities. We developed a novel mathematical technique and determined kinetics in presence of ACh. The results suggest occupancy of ACh of one binding site increases dissociation rate of antagonist from the other site. We incorporated all of these rate constants into a computer simulation of a comprehensive 11-state kinetic model. There was excellent agreement (without curve fitting) between simulated and experimental currents, thereby establishing the significance of each rate constant and the high accuracy and internal consistency of our system.; We examined the effect of a point mutation (alphaY198F) on the kinetics of (+)-tubocurarine inhibition. We also examined temperature dependence (25°C vs. 37°C) of agonist-induced nAChR activation and of kinetics of competitive antagonism. Comparable experiments have not been performed previously for any ligand-gated ion channel. The binding of both agonists and antagonists were primarily enthalpy-driven. The enthalpy- and entropy-drives of agonists correlated linearly with their structural size. "Competitive" antagonists do not compete with ACh for the same site. Instead, they bind at another site and sterically hinder access of ACh to its binding site, yielding the same apparent effect as classical competitive antagonists. Antagonists differ in the strength of their conserved interactions with the receptor. Their binding can be represented by two antagonist-specific energy wells: (1) distinct diffusional barrier encountered along the pathway; (2) conformational change (i.e., contraction of binding site) that stabilizes docking of antagonist (induced fit). In summary, this dissertation furthers the understanding of the dynamic molecular nature of ligand-receptor interactions and provides insight into clinical actions of muscle relaxants.