Simultaneous single-molecule optical and electrical measurements of ion channel ligand binding and pore gating

项目总结 配体门控离子通道(LGIC)是一种分子传感器，它将配体结合的化学能转化为 通过离子通量通过通道孔的电脉冲。LGIC是突触传递所必需的 整个神经系统以及许多其他基本生理过程中的细胞信号 比如视觉、嗅觉、运动控制和心率，仅举几例。他们也是主要的毒品目标，因为 调节频道行为可以用来抵消一系列的痛苦，例如焦虑，成瘾， 疼痛、肌肉损伤等。通道孔的门控(打开/关闭)在配体结合时启动， 通常到不同子单元或域中的多个站点。尽管在理解三个方面取得了重大进展- LGIC的维度结构，在我们对这些领域如何 参与塑造化学结合能被传递到离子门控的事件序列 毛孔。弥合这一差距的一个主要障碍是，需要单分子方法来解决随机 绑定和门控事件仅报告绑定刺激或门控响应，而不是同时报告 需要了解完整的刺激-反应途径。为了克服这个障碍，我将使用一种创新的 微镜全内反射荧光(MMTIRF)单分子成像与光学结合 跟踪荧光标记配体的单个结合事件，同时记录离子传导 用传统的膜片钳技术在切除的膜片中通过单通道。目标是 这项建议的目的是建立这种联合方法激活环核苷酸门控的可行性 (CNG)对视觉和嗅觉至关重要的通道。其基本原理是，MMTIRF和 单通道记录将在多个不同的结合事件之间实现直接实验关联 电域和电门控响应。完成这一目标将1)建立一种便捷的方法 在单分子分辨率下探索LGIC中的完整刺激-反应通路，以及2)确定程度 结合一个或两个环核苷酸的能量被传递到孔门的打开。这个 拟议的研究具有重要意义，因为它将使探索事件动态序列的研究成为可能 控制多个区域中的化学结合能向离子孔门控的转换，这是一个过程 在许多情况下，人们对此知之甚少。这项提案中制定的方法将对 了解配体驱动通道活动的动态事件以及它们之间的连接点 静态结构快照，从而将构成离子通道领域向前迈出的一大步。此外， 它将直接影响对药物调节通道行为的机制的理解，这将 促进新型治疗调节剂的合理设计。

PROJECT SUMMARY Ligand-gated ion channels (LGICs) are molecular sensors that convert the chemical energy of ligand binding to electrical impulses via ion flux through the channel pore. LGICs are essential for synaptic transmission throughout the nervous system as well as cellular signaling in many other fundamental physiological processes such as vision, olfaction, motor control and heart rate to name just a few. They are also major drug targets as modulating channel behavior can be used to counteract a wide range of afflictions such as anxiety, addiction, pain, muscle impairment, etc. Gating (opening/closing) of the channel pore is initiated upon binding of ligands, often to multiple sites in distinct subunits or domains. Despite significant progress in understanding the 3- dimensional structure of LGICs, there remains a critical gap in our understanding of how these domains participate to shape the sequence of events by which chemical binding energy is transduced to gating of the ion pore. A major barrier to bridging this gap is that the single-molecule methods needed to resolve the stochastic binding and gating events only report on either the binding stimulus or the gating response, but not both as required to understand the full stimulus-response pathway. To overcome this barrier, I will use an innovative combination of micro-mirror total internal reflection fluorescence (mmTIRF) single-molecule imaging to optically track individual binding events for a fluorescently labeled ligand while simultaneously recording ion conduction through single channels in excised membrane patches with conventional patch clamp techniques. The objective of this proposal is to establish the feasibility of this combined approach for activation of cyclic nucleotide gated (CNG) channels critical for visual and olfactory sensation. The rationale is that the combination of mmTIRF and single-channel recording will enable direct experimental correlation between distinct binding events at multiple domains and the electrical gating response. Completion of this objective will 1) establish a facile approach for probing the full stimulus-response pathway in LGICs at single-molecule resolution, and 2) determine the degree to which energy from binding one or two cyclic nucleotides is transduced to opening of the pore gate. The proposed research is significant because it will enable studies that probe the dynamic sequence of events governing the transduction of chemical binding energy in multiple domains to gating of the ion pore, a process which in many cases is only poorly understood. The approach developed in this proposal will be invaluable to understanding the dynamic events by which ligands drive channel activity and which connect the dots between static structural snapshots, and thereby will constitute a major step forward for the ion channel field. Furthermore, it will have direct bearing on understanding the mechanisms of drugs that modulate channel behavior, which will facilitate the rational design of novel therapeutic modulators.