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

项目摘要 缺血性中风可导致长期认知损害，导致近一半的人行动能力下降。 中风幸存者在缺血性事件期间和之前发生的脑组织损伤通常在很大程度上部分是由缺血性事件引起的。 因为严重的局部组织酸中毒虽然酸中毒导致组织损伤的分子机制是 质子门控离子通道被认为起作用，但这在很大程度上是未知的。一种新的质子门控氯离子通道 最近被鉴定为以前未表征的基因TMEM 206，现在通常被称为 质子激活氯离子通道（PAC）虽然最近的研究表明PAC在酸诱导的细胞凋亡中起作用， 死亡，不存在通道的质子激活氯电流的分子理由。这项建议 将整合电生理学，生物化学和高分辨率结构实验，以阐明 以结构为基础的机制，管理PAC的功能。为了实现这一目标，我将首先获得高- 用单粒子冷冻电子显微术解析PAC的静止和活动功能状态的结构 （冷冻-EM）。这些结构将提供基本的见解的架构，化学计量，和独特的 PAC的蛋白质折叠这些信息也将扩大我们的知识周围的物理和化学 质子门控离子通道的性质，以及广泛地告知离子通道的结构/功能关系， 渠道然后，我将确定PAC的pH依赖性机制和孔的分子基础 通过使用结构定向诱变和电生理学方法探测PAC的功能， 实验这些实验将建立PAC的分子结构和生理学之间的联系。 最终，该提案将定义PAC功能的基于结构的生化机制，这将奠定 为未来的研究奠定基础，并可能为治疗剂的开发提供信息，以减轻神经元的 缺血性事件中的损伤。 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 扩大我的知识基础和技术技能，在离子通道，膜蛋白生物化学， 电生理学和冷冻电镜对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，Eric Gouaux博士是配体门控离子通道的专家，也是 膜蛋白结构生物学Gouaux博士是OHSU Vollum研究所的高级科学家， 电生理发电站，是太平洋西北部三个国家冷冻EM中心之一的所在地 冷冻EM中心（PNCC）。总的来说，该项目不仅将阐明对小说的批评见解， 质子激活通道，但也将为我提供必要的知识，生物物理工具包， 我需要的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.