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

离子通道配体结合和孔门控的同时单分子光学和电学测量

• 批准号: 10575611
• 负责人: Marcel Paz Goldschen-Ohm
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10575611
• 关键词: Anxiety; Automobile Driving; Behavior; Binding; Cardiac; Chemicals; Cyclic GMP; Cyclic Nucleotides; DNA Sequence Alteration; Data; Disease; Dissection; Drug Targeting; Electrophysiology (science); Ensure; Esthesia; Event; Fluorescence; Fluorescence Microscopy; Geometry; Heart Rate; Image; Impairment; Individual; Ion Channel; Ion Channel Gating; Ions; Label; Ligand Binding; Ligands; Measures; Membrane; Methods; Molecular; Muscle; Muscle Contraction; Names; Nature; Nervous system structure; Neurologic; Optics; Pain; Patch-Clamp Techniques; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Physiological Processes; Positioning Attribute; Preparation; Probability; Process; Reporting; Research; Resolution; Response to stimulus physiology; Sensory; Shapes; Signal Transduction; Site; Smell Perception; Stimulus; Surface; Synaptic Transmission; System; Techniques; Transducers; Vision; Visual; addiction; analog; chemical binding; confocal imaging; cyclic-nucleotide gated ion channels; electrical measurement; fluorescence imaging; imaging modality; innovation; micromanipulator; molecular imaging; motor control; nano; nanometer; novel therapeutics; patch clamp; pressure; rational design; response; sensor; single molecule; therapy design; three dimensional structure; tool; voltage; voltage gated channel

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.

项目摘要 配体门控离子通道（LGIC）是分子传感器，将配体结合的化学能转化为 通过通道孔通过离子通量进行电脉冲。 LGIC对于突触传输至关重要 在整个神经系统以及许多其他基本生理过程中的细胞信号传导 例如视觉，嗅觉，运动控制和心率仅举几例。它们也是主要的药物目标 调节通道行为可用于抵消各种痛苦，例如焦虑，成瘾， 疼痛，肌肉障碍等。在结合配体时，启动了通道孔的门控（打开/关闭）， 通常在不同的亚基或域中的多个站点。尽管在理解3-的情况下取得了重大进展 LGIC的维度结构，我们对这些领域的理解中仍然存在一个危险的差距 参与以塑造将化学结合能转导为离子门控的事件的序列 毛孔。弥合此差距的主要障碍是解决随机的单分子方法 结合和门控事件仅报告结合刺激或门控反应 需要了解完整的刺激响应途径。为了克服这个障碍，我将使用创新 微晶格总内反射荧光（MMTIRF）单分子成像的组合与光学成像 跟踪荧光标记配体的单个结合事件，同时记录离子传导 通过使用常规贴片夹技术切除的膜贴片中的单个通道。目标 该建议的是建立这种联合方法激活环状核​​苷酸门的可行性 （CNG）通道对视觉和嗅觉至关重要。理由是mmtirf和 单通道记录将使多个不同的结合事件之间的直接实验相关 域和电控响应。完成此目标的完成1）建立一种便利的方法 在单分子分辨率下探测LGICS中的全部刺激 - 反应途径，并确定程度 从结合一个或两个环状核苷酸的能量转换为孔门的打开。这 拟议的研究很重要，因为它将实现探测事件动态序列的研究 管理多个结构域中化学结合能的转导向离子孔的门控，这一过程 在许多情况下，只有很少的理解。在本提案中开发的方法对于 了解配体驱动通道活动的动态事件以及连接点之间的点 静态结构快照将构成离子通道场的重要一步。此外， 它将直接取决于理解调节通道行为的药物的机制，这将 促进新型治疗调节剂的合理设计。