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

项目总结 离子通道在结合不同亚基或结构域的多个配体时的激活对突触是必不可少的 传输和细胞信号。尽管最近在理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着根本的差距， 多个结合位点和结构域协同打开和关闭通道孔。架设桥梁的主要障碍 这一差距是，来自多个渠道的集合平均约束性措施同时遮挡了对 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。至 克服这个障碍，我将使用我的实验室开发的创新的单分子荧光方法 结合我之前在实现光学跟踪的零模波导纳米光子阵列方面的专业知识 每个单独的绑定步骤。该提案的目标是确定1)能源环境 驱动环核苷酸门控(CNG)通道激活的步进结合事件序列 视觉和嗅觉，以及2)苯二氮卓类药物(BZD)对GABAA受体的调节，BZD是一种 当今最广泛使用的精神药物。其基本原理是对个体绑定进行光学跟踪 作为这些通道运行的化学刺激的事件将使序列的确定成为可能 指不同的能量事件，必须至少部分发生在气孔打开之前，因此很难测量 通过电生理学方法。具体目标是：1)建立顺应时代的能源格局 在CNG通道处结合；2)利用每个不同的结合步骤来量化CNG通道开放的可能性， 这将检验现有不同的模型预测；3)开发CNG通道激活的机制模型 这解释了每个不同的结合步骤；4)确定BZD结合或序列的能量格局 激动剂与GABAA受体的结合，以及5)确定BZD是否改变不同的激动剂结合步骤。 拟议的研究具有重要意义，因为它将为理解 控制这些通道中配体驱动行为的事件的动态序列，目前仅保留 人们对此知之甚少。作为量化基准，这些结果将立即产生积极影响 计算、结构和功能研究，旨在揭示观察到的 能量的变化。最终，了解通道激活过程中的完整事件序列是至关重要的 不仅是为了增进我们对离子通道机制的基础知识，也是为了促进发展 针对激活途径中不同步骤的治疗。从长远来看，这种认识将使理性 设计新的治疗方法以改善治疗结果和生活质量。

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.