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

项目摘要 配体门控离子通道（LGIC）是分子传感器，其将配体结合的化学能转化为 通过离子流通过通道孔的电脉冲。LGIC对突触传递至关重要 以及许多其他基本生理过程中的细胞信号 例如视觉、嗅觉、运动控制和心率。它们也是主要的药物靶点， 调节通道行为可用于抵消多种痛苦，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， 通常在不同的亚基或结构域中的多个位点。尽管在理解3- LGIC的三维结构，在我们理解这些领域如何 参与形成将化学结合能转换为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，单分子方法需要解决的随机 结合和门控事件仅报告结合刺激或门控反应，但不同时报告两者， 了解完整的刺激反应途径。为了克服这一障碍，我将使用一种创新的 微镜全内反射荧光（mmTIRF）单分子成像与光学成像的组合 跟踪荧光标记配体的单个结合事件，同时记录离子传导 通过传统膜片钳技术在切除的膜膜片中的单通道。客观 该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)视觉和嗅觉的关键通道基本原理是mmTIRF和 单通道记录将使得能够在多个不同的结合事件之间进行直接的实验关联。 域和电门控响应。完成这一目标将：1）建立一个简便的方法， 以单分子分辨率探测LGIC中的完整刺激-反应途径，以及2）确定 来自结合一个或两个环核苷酸的能量被传递到该孔中以打开孔门。的 拟议的研究是重要的，因为它将使研究，探讨动态序列的事件 控制多个域中的化学结合能的转导到离子孔的门控， 这在很多情况下只是知之甚少。本提案中提出的方法将非常宝贵， 了解配体驱动通道活动的动态事件，以及连接 静态结构快照，从而将构成离子通道领域的重大进步。此外，委员会认为， 它将直接关系到对调节通道行为的药物机制的理解， 有助于合理设计新的治疗调节剂。

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.