Mechanisms of stepwise activation and drug-modulation in ligand-gated ion channels.

配体门控离子通道的逐步激活和药物调节机制。

• 批准号: 10710047
• 负责人: Marcel Paz Goldschen-Ohm
• 金额: $ 32.74万
• 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: 2022-09-26 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10710047
• 关键词: Address; Affect; Agonist; Anxiety; Behavior; Benchmarking; Benzodiazepine Receptor; Benzodiazepines; Binding; Binding Sites; Cells; Chemicals; Color blindness; Coupling; Cyclic GMP; Cysteine; DNA Sequence Alteration; Diameter; Disparate; Disulfides; Drug Modulation; Drug Targeting; Electrophysiology (science); Esthesia; Event; Fluorescence; Foundations; Goals; Image; Imaging Techniques; Immobilization; Impairment; Individual; Ion Channel; Ion Channel Gating; Ions; Kinetics; Knowledge; Label; Left; Ligand Binding; Ligands; Literature; Measures; Mediator; Membrane Proteins; Methods; Modeling; Molecular Conformation; Muscle; Nature; Optics; Pain; Pathway interactions; Pharmaceutical Preparations; Psychotropic Drugs; Quality of life; Reporting; Research; Role; Shapes; Signal Transduction; Site; Stimulus; Synaptic Transmission; Testing; Treatment outcome; Vesicle; Visual; Work; addiction; analog; cyclic-nucleotide gated ion channels; detection limit; fluorescence imaging; improved; innovation; molecular imaging; nanophotonic; nanovesicle; nervous system disorder; novel therapeutics; pharmacologic; predictive modeling; preference; rational design; receptor; receptor binding; single molecule; targeted treatment; therapeutic target; therapy development; three dimensional structure; waveguide

PROJECT SUMMARY Activation of ion channels upon binding multiple ligands at distinct subunits or domains is essential for synaptic transmission and cellular signaling. Despite recent advances in understanding their 3-dimensional structure, there remains for many channels a fundamental gap in our understanding of the sequence of events by which multiple binding sites and domains coordinate to open and close the channel pore. A major barrier to bridging this gap is that ensemble-averaged binding measures from many channels at once occlude observation of the distinct asynchronous binding steps that underlie the sequence of binding events at each individual channel. To overcome this barrier, I will use innovative single-molecule fluorescence methods developed in my lab in combination with my prior expertise with zero-mode waveguide nanophotonic arrays that enable optical tracking of each individual binding step. The objective of this proposal is to determine the energy landscape for 1) the sequence of stepwise binding events that drive activation of cyclic nucleotide gated (CNG) channels critical for visual and olfactory sensation, and 2) modulation of GABAA receptors by benzodiazepines (BZDs), one of the most widely prescribed psychotropic drugs today. The rationale is that optical tracking of individual binding events that are the chemical stimuli by which these channels operate will enable determination of the sequence of distinct energetic events that must at least partially occur prior to pore opening and thus are difficult to measure with electrophysiological approaches. The specific aims will: 1) Establish the energy landscape for sequential binding at a CNG channel; 2) Quantify the likelihood of CNG channel opening with each distinct binding step, which will test existing disparate model predictions; 3) Develop a mechanistic model for CNG channel activation that accounts for each distinct binding step; 4) Determine the energy landscape for BZD-binding or sequential agonist binding at GABAA receptors, and 5) Establish whether or not BZDs alter distinct agonist binding steps. The proposed research is significant because it will provide a necessary foundation for understanding the dynamic sequence of events governing ligand-driven behavior in these channels, which currently remain only poorly understood. The results will have an immediate positive impact as a quantitative benchmark for computational, structural, and functional studies aimed at uncovering the physical basis for the observed changes in energy. Ultimately, understanding the full sequence of events during channel activation is essential not only to advance our fundamental knowledge of ion channel mechanisms, but also to facilitate development of therapies targeting distinct steps in the activation pathway. Long-term, this knowledge will enable the rational design of new therapies to improve treatment outcomes and quality of life.

项目摘要 在不同亚基或域上结合多个配体时，激活离子通道对于突触是必不可少的 传递和细胞信号传导。尽管最近在理解其三维结构方面取得了进步，但 许多渠道仍存在我们对事件顺序的理解的根本差距 多个结合位点和域坐标以打开和关闭通道孔。桥接的主要障碍 这一差距是从许多通道的合奏平均结合度量立即遮住了观察 不同的异步结合步骤是每个通道处结合事件序列的基础。到 克服这个障碍，我将使用在我的实验室中开发的创新单分子荧光方法 结合我先前的专业知识与零模式波导纳米光阵列，可实现光学跟踪 每个单独的绑定步骤。该提议的目的是确定1） 驱动环环核苷酸门控（CNG）通道激活的逐步结合事件的序列 视觉和嗅觉，以及2）苯二氮卓类（BZDS）对GABAA受体的调节，其中之一 如今，规定的精神药物最广泛。理由是单个绑定的光学跟踪 这些通道运行的化学刺激的事件将确定序列 在孔口打开之前至少必须部分发生的独特的充满活力的事件，因此很难测量 使用电生理方法。具体目的将：1）建立顺序的能量格局 在CNG通道上结合； 2）用每个独特的结合步骤量化CNG通道打开的可能性， 将测试现有的不同模型预测； 3）开发一个用于CNG通道激活的机械模型 这说明了每个独特的绑定步骤； 4）确定BZD结合或顺序的能量景观 在GABAA受体处的激动剂结合，5）确定BZD是否改变了不同的激动剂结合步骤。 拟议的研究很重要，因为它将为理解 这些通道中控制配体驱动行为的事件的动态序列，目前仅保留 理解不佳。结果将立即产生积极影响，作为定量基准 计算，结构和功能研究旨在发现观察到的物理基础 能量变化。最终，了解通道激活期间的全部事件是必不可少的 不仅要促进我们对离子渠道机制的基本知识，而且还促进发展 靶向激活途径中不同步骤的疗法。长期，这些知识将使理性 设计新疗法以改善治疗结果和生活质量。