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 在不同的亚基或结构域结合多个配体后，离子通道的激活对于突触传递是必需的。 传输和蜂窝信号。尽管最近在了解它们的三维结构方面取得了进展， 对于许多渠道来说，我们对事件顺序的理解仍然存在根本性的差距， 多个结合位点和结构域协调以打开和关闭通道孔。桥接的主要障碍 这一差距是，来自许多通道的整体平均结合测量同时阻碍了对 不同的异步绑定步骤，这些步骤是每个单独通道上绑定事件序列的基础。到 为了克服这一障碍，我将使用我实验室开发的创新单分子荧光方法， 结合我之前在零模式波导纳米光子阵列方面的专业知识， 每一个单独的绑定步骤。本提案的目的是确定以下方面的能源前景：1） 一系列逐步结合事件，驱动环核苷酸门控（CNG）通道的激活，这些通道对于 视觉和嗅觉，和2）苯二氮卓类（BZD）对GABAA受体的调节， 最常用的精神药物基本原理是，单个结合的光学跟踪 作为这些通道运作的化学刺激的事件将使得能够确定序列 必须至少部分地在孔隙打开之前发生的不同的能量事件，因此难以测量 用电生理学的方法。具体目标是：1）建立能源格局， 在CNG通道处的结合; 2）用每个不同的结合步骤量化CNG通道开放的可能性， 这将测试现有的不同模型预测; 3）开发CNG通道激活的机理模型 4）确定BZD结合或顺序结合的能量景观; GABAA受体的激动剂结合，和5）确定BZD是否改变不同的激动剂结合步骤。 这项研究具有重要的意义，因为它将为理解 这些通道中控制配体驱动行为的动态事件序列，目前仅 不太了解。结果将立即产生积极影响，作为量化基准， 计算，结构和功能研究，旨在揭示所观察到的物理基础 能量的变化。最后，了解通道激活过程中事件的全部顺序是至关重要的 不仅可以提高我们对离子通道机制的基础知识， 针对激活途径中不同步骤的治疗。从长远来看，这些知识将使理性 设计新的治疗方法，以改善治疗效果和生活质量。