The molecular architecture and mechanism of the Proton Activated Chloride (PAC) Channel.

项目摘要 缺血性中风会导致长期的认知损害，导致近一半的人行动不便 中风幸存者。在缺血事件期间和之前发生的脑组织损伤通常是部分 由于局部组织严重酸中毒。而酸中毒如何导致组织损伤的分子机制是 人们认为，质子门控离子通道在其中发挥了作用，这一点在很大程度上不为人知。一种新型的质子门控氯离子通道 最近被确定为以前未描述的基因TMEM206，现在通常被称为 质子活化氯(PAC)通道。虽然最近的研究表明PAC与酸诱导的细胞有关 死亡，不存在该通道的质子激活的氯电流的分子理由。这项建议 将结合电生理、生化和高分辨率结构实验来阐明 管理政治行动委员会职能的基于结构的机制。为了支持这一目标，我将首先获得- PAC静息和活性功能状态的单粒子冷冻电子显微镜分辨结构 (冷冻-EM)。这些结构将提供对体系结构、化学计量和独一无二的基本见解 PAC的蛋白质折叠。这些信息还将扩大我们对物理和化学方面的知识 质子门控离子通道的性质，以及广泛地告知离子的结构/功能关系 频道。然后我将确定PAC的pH依赖机制和毛孔的分子基础 用结构定向突变和电生理方法研究PAC的功能 实验。这些实验将在PAC的分子结构和生理之间建立联系。 最终，这项提议将为PAC的功能定义基于结构的生化机制，这将为 为未来的研究奠定基础，并可能为治疗药物的开发提供信息，以减轻神经元 在缺血事件中的损害。 作为一名神经科学研究生，他的目标是成为一名独立的学术科学家 将研究神经系统配体门控离子通道的结构/功能，该项目将直接 拓展我在离子通道、膜蛋白生物化学、 电生理学和冷冻电磁场。对PAC的分子结构和作用机理的研究将是 在Eric Gouaux博士的指导下进行研究，他是配体门控离子通道方面的专家，也是 膜蛋白结构生物学。古奥博士是俄亥俄州立大学沃勒姆研究所的资深科学家。 太平洋西北部三个国家冷冻-EM中心之一的电生理学中心 冷冻-EM中心(PNCC)。综上所述，该项目不仅将阐明对一部小说的批判性见解 质子激活的通道，但也将为我提供必要的知识、生物物理工具包和 我需要专业技能来实现我的长期职业目标。

Project Summary Ischemic strokes can cause long-term cognitive damage, leading to reduced mobility in nearly half of stroke survivors. Brain tissue damage that occurs during and preceding an ischemic event is often largely in part due to severe local tissue acidosis. While the molecular mechanism of how acidosis leads to tissue damage is largely unknown, proton-gated ion channels are thought to play a role. A novel proton-gated chloride channel has been recently identified as the previously uncharacterized gene, TMEM206, now commonly referred to as the Proton-Activated Chloride (PAC) Channel. While recent studies have implicated PAC in acid-induced cell death, there exists no molecular justification of the channel’s proton-activated chloride currents. This proposal will integrate electrophysiological, biochemical, and high-resolution structural experiments to elucidate the structure-based mechanisms that govern the function of PAC. In support of this goal, I will first obtain the high- resolution structures of PAC’s resting and active functional state using single particle cryo-electron microscopy (Cryo-EM). These structures will provide fundamental insights into the architecture, stoichiometry, and unique protein folding of PAC. This information will also expand our knowledge surrounding the physical and chemical properties of proton-gated ion channels, as well as broadly inform the structure/function relationship of ion channels. I will then ascertain the molecular underpinnings of PAC’s pH-dependent mechanism and pore properties by probing PAC’s function using structure-directed mutagenesis and electrophysiological experiments. These experiments will establish a link between the molecular architecture and physiology of PAC. Ultimately, this proposal will define structure-based, biochemical mechanisms for PAC’s function, which will lay the foundation for future studies and may inform the development of therapeutic agents to mitigate neuronal damage in ischemic events. As a neuroscience graduate student whose goal is to become an independent academic scientist that will study the structure/function of ligand-gated ion channels of the nervous system, this project will directly expand my knowledge base and technical skillset in ion channels, membrane protein biochemistry, electrophysiology, and cryo-EM. The study into the molecular architecture and mechanism of PAC will be pursued under the mentorship of Dr. Eric Gouaux, an expert in ligand-gated ion channels and leader in membrane protein structural biology. Dr. Gouaux is a senior scientist at the Vollum Institute at OHSU, an electrophysiology powerhouse that is home to one of three national centers for Cryo-EM, Pacific Northwest Center for Cryo-EM (PNCC). Taken together, the project will not only illuminate critical insights into a novel proton-activated channel, but will also provide me with the necessary knowledge, biophysical tool-kit, and professional skills I need to achieve my long-term career goal.