Marabelli Alessandro, Lape Remigijus, Sivilotti Lucia
Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, England, UK.
Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, England, UK
J Gen Physiol. 2015 Jan;145(1):23-45. doi: 10.1085/jgp.201411234.
Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.
原核生物通道，如菊欧文氏菌配体门控离子通道（ELIC）和紫球藻配体门控离子通道，为五聚体配体门控离子通道家族提供了关键的结构信息，该家族包括烟碱型乙酰胆碱受体。ELIC是一种来自菊欧文氏菌的阳离子通道，因其高电导率特别适合单通道记录。在此，我们报告了在人胚肾293细胞中表达的ELIC通道的动力学特性。通过将动力学方案直接最大似然拟合到理想化数据，分析了在-60 mV下，全激动剂丙胺（0.5 - 50 mM）在外侧向外膜片中引发的单通道电流。测试了几种机制，并通过将最佳拟合的预测结果与实验数据的可观测特征进行比较来判断其适用性。这些特征包括开放/关闭时间分布以及通过快速θ管施加（50 - 600 ms，1 - 50 mM，-100 mV）引发的宏观丙胺激活电流的时间进程。相关的真核生物通道，如甘氨酸和烟碱型受体，在完全结合配体时会高效地开放到单一开放状态，通过一个预开放中间体实现。对其激活的最简单合适描述，即“翻转”模型，假设在高激动剂浓度下协同转变为单一中间体状态。相比之下，在饱和丙胺条件下ELIC的开放时间分布显示出多个成分。因此，完全结合配体的通道必定可以进入不止一种开放状态。“准备好的”模型允许从多个完全结合配体的中间体开放。这种类型模型的最佳拟合表明，当至少两个且可能三个激动剂分子与通道结合时，ELIC达到最大开放概率（99%）。完全结合配体的通道开放的总体效能约为102（α1β甘氨酸通道约为20）。随着通道激活，对激动剂的微观亲和力增加，从静息状态的7 mM增加到部分激活中间体状态的0.15 mM。
