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

质子活化氯化物 (PAC) 通道的分子结构和机制。

基本信息

批准号：
10311483
负责人：
Makayla Freitas
金额：
$ 0.01万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-28 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10311483
关键词：
ASIC channel Acidosis Acids Address Amino Acids Anions Architecture Biochemical Biological Assay Biological Process Biophysics Brain C-terminal Cell Death Cells Cerebral Ischemia Characteristics Chloride Channels Chloride Ion Chlorides Code Cognitive Cryoelectron Microscopy Data Development Drug Targeting Electrophysiology (science)Environment Event Family Foundations Future Genes Goals Home Homeostasis Institutes Investigation Ion Channel Ion Channel Gating Ions Ischemia Ischemic Brain Injury Ischemic Stroke Knock-out Knowledge Ligands Link Mediating Membrane Proteins Mentorship Modeling Molecular Mutagenesis Mutate Mutation N-terminal Nervous system structure Neuraxis Neuronal Injury Neurons Neurosciences Pacific Northwest Physiological Physiological Processes Physiology Play Prevention Property Protein Biochemistry Protons Resolution Rest Role Scientist Senior Scientist Shapes Sodium Stroke Structure Structure-Activity Relationship Swelling Therapeutic Agents Tissues Translating Traumatic Brain Injury alpha helix base biophysical properties brain tissue career cell growth regulation chemical property desensitization design experimental study extracellular graduate student human tissue insight knowledge base millisecond mouse model mutant neuron loss novel novel therapeutics particle physical property protein folding response skills stoichiometry stroke survivor structural biology therapeutic development therapeutic target

项目摘要

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.

项目摘要缺血性中风会导致长期认知损害，从而导致几乎一半的流动性降低中风存活。脑组织损伤在缺血事件之前和之前发生的脑组织损伤通常很大。由于严重的局部组织酸中毒。而酸中毒如何导致组织损伤的分子机制是人们认为，质子门控的离子通道在很大程度上扮演了角色。一种新型的质子门控氯化物通道最近已被鉴定为先前未表征的基因TMEM206，现在通常称为质子活化的氯化物（PAC）通道。虽然最近的研究已经在酸诱导的细胞中实施了PAC 死亡，没有任何分子依据对通道的质子激活的氯水流。这个建议将整合电生理，生化和高分辨率结构实验，以阐明控制PAC功能的基于结构的机制。为了支持这个目标，我将首先获得高级使用单个粒子冷冻电子显微镜的PAC静止和主动功能状态的分辨率结构（Cryo-Em）。这些结构将为建筑，化学计量和独特的建筑提供基本见解 PAC的蛋白质折叠。这些信息还将扩大我们围绕物理和化学的知识质子门控离子通道的特性，并广泛地告知离子的结构/功能关系频道。然后，我将确定PAC pH依赖性机理和孔的分子基础通过使用结构定向的诱变和电生理探测PAC的功能，属性实验。这些实验将在PAC的分子结构与生理学之间建立联系。最终，该建议将定义基于结构的PAC功能的生化机制未来研究的基础，并可能告知治疗剂的发展以减轻神经元缺血事件的损害。作为一名神经科学研究生，其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，该项目将直接在离子渠道，膜蛋白生物化学中扩展我的知识库和技术技能，电生理学和冷冻EM。 PAC的分子结构和机制的研究将是在配体门控离子渠道的专家埃里克·古阿克斯（Eric Gouaux）的心态下追求膜蛋白结构生物学。 Gouaux博士是OHSU Vollum Institute的高级科学家，电生理电力公司是太平洋西北部三个国家中心之一的所在地之一冷冻EM（PNCC）中心。综上所述，该项目不仅会阐明对小说的批判性见解质子激活的渠道，但也将为我提供必要的知识，生物物理工具套件和我需要实现长期职业目标所需的专业技能。