The dissociation of acetylcholine from open nicotinic receptor channels
The dissociation of acetylcholine from open nicotinic receptor channels
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DOI：
10.1073/pnas.251402498
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发表时间：
2001-11-20
影响因子：
11.1
通讯作者：
Auerbach, A
中科院分区：
文献类型：
--
作者：
Grosman, C;Auerbach, A
配体门控离子通道在开放状态下比在关闭状态下以更高的亲和力结合激动剂。这种增加的亲和力的动力学基础仍然未知，因为即使可以合理确定地估计激动剂与封闭受体结合和解离的速率常数，开放受体中结合步骤的动力学已被证明是难以捉摸的。为了能够测量开放肌肉烟碱受体的激动剂解离速率常数，我们通过设计一些加速开放和减慢关闭但保持配体结合特性不变的突变，增加了配体从开放状态解离的可能性。在极低浓度的乙酰胆碱 (ACh) 下对野生型和突变体构建体进行单通道膜片钳记录。分析单个通道激活的持续时间，假设完全配体（双配体）受体开口的“爆发”可以通过配体从关闭或开放状态解离（随后快速关闭）或通过脱敏来终止。该分析表明，ACh 从双配体开放受体解离的时间约为 24 s(-1)，即比从双配体封闭受体慢大约 2,500 倍。这种变化单独存在，而没有伴随与开放状态的关联速率常数的变化，定量地解释了开放通道对ACh的平衡亲和力的增加。此外，结果预测，脱敏和 ACh 从开放状态解离经常终止自然发生的功能获得突变体（导致慢通道先天性肌无力）的开放爆发，因此将显着影响这些疾病条件下终板电流衰减的时间进程。
Ligand-gated ion channels bind agonists with higher affinity in the open than in the closed state. The kinetic basis of this increased affinity has remained unknown, because even though the rate constants of agonist association to and dissociation from closed receptors can be estimated with reasonable certainty, the kinetics of the binding steps in open receptors have proven to be elusive. To be able to measure the agonist-dissociation rate constant from open muscle nicotinic receptors, we increased the probability of ligand unbinding from the open state by engineering a number of mutations that speed up opening and slow down closing but leave the ligand-binding properties unchanged. Single-channel patch-clamp recordings from the wild-type and mutant constructs were performed at very low concentrations of acetylcholine (ACh). The durations of individual channel activations were analyzed assuming that "bursts" of fully liganded (diliganded) receptor openings can be terminated by ligand dissociation from the closed or open state (followed by fast closure) or by desensitization. This analysis revealed that ACh dissociates from diliganded open receptors at approximate to 24 s(-1), that is, approximate to2,500 times more slowly than from diliganded closed receptors. This change alone without a concomitant change in the association rate constant to the open state quantitatively accounts for the increased equilibrium affinity of the open channel for ACh. Also, the results predict that both desensitization and ACh dissociation from the open state frequently terminate bursts of openings in naturally occurring gain-of-function mutants (which cause slow-channel congenital myasthenia) and therefore would contribute significantly to the time course of the endplate current decay in these disease conditions.