Structural and Functional Studies on Proton-activated Chloride (PAC) Channel

质子激活氯离子 (PAC) 通道的结构和功能研究

• 批准号: 10681494
• 负责人: Zheng Ruan
• 金额: $ 10.93万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10681494
• 关键词: Acidosis; Acids; Adopted; Anions; Antibodies; Antibody Formation; Architecture; Award; Benzoic Acids; Binding; Binding Sites; Biochemical; Brain; Brain Injuries; Cell Death Induction; Cell Line; Cells; Cerebrum; Cessation of life; Chloride Channels; Chlorides; Computing Methodologies; Cryoelectron Microscopy; Development; Disease; Electrophysiology (science); Extracellular Domain; Foundations; Future; Genes; Goals; Human; Immunize; Injury; Intervention; Investigation; Ions; Ischemic Stroke; Knowledge; Link; Lipids; Mediating; Membrane Proteins; Mentors; Metabolic; Molecular; Molecular Conformation; Monoclonal Antibodies; Mouse Protein; Mus; Mutation; Neurons; Niflumic Acid; Orthologous Gene; Outcome; Pathologic; Pathologic Processes; Pathway interactions; Patients; Pharmacologic Substance; Phase; Physiological; Play; Post-Translational Protein Processing; Postdoctoral Fellow; Probability; Procedures; Property; Proteins; Protons; RNA Splicing; Recombinants; Research; Research Personnel; Rest; Role; Services; Severities; Solid; Structure; Supervision; Swelling; System; Techniques; Therapeutic; Time; Tissues; Titrations; Toxic effect; Training; United States; Variant; Work; channel blockers; design; disability; effective therapy; experimental study; extracellular; improved; inhibitor; insight; mutant; nanodisk; neurotoxicity; novel; particle; patch clamp; pharmacologic; pregnenolone sulfate; protein purification; protein structure; response; screening; sensor; small molecule; stoichiometry; treatment strategy; voltage

ABSTRACT Ischemic stroke is one of the leading causes of disability and death in the United States. Acid accumulation in the brain during ischemic stroke causes neurotoxicity and irreversible tissue damage. Understanding the factors that contribute to acid-induced cell death during ischemic stroke is thus critical to define the pathological process and develop effective treatment strategies. The proton-activated chloride (PAC) channel (also known as ASOR or PAORAC) is a recently discovered cellular pH-sensor that plays a critical role in determining the outcome of brain damage after ischemic stroke. Under acidic conditions, the activation of PAC allows an influx of chloride current into the neuron which further causes cell swelling and death. In 2019, the PAC gene was cloned by two independent groups and was found to be a novel chloride channel. In 2020, I revealed the first near-atomic cryo-EM structures of the human PAC channel at two different conformational states, including an apo state and a proton-bound non-conducting state. Our study provided a wealth of information about channel stoichiometry, domain architecture, and anion selectivity mechanisms of PAC. While promising progress has been made towards understanding the function of this channel, a complete picture of how PAC responds to environmental acidification is still obscure due to the limited knowledge about the pH- sensor and the lack of an open state structure. Likewise, although the PAC current is sensitive to several non- specific chloride channel blockers, their inhibition mechanisms are unexplored. The long-term objective of this research is to unveil the molecular principles underlying PAC channel function in both physiological and pathological conditions, and to develop specific compounds that could be used to mitigate the effect of ischemic stroke in patients. In this K99/R00 proposal, will carry out a comprehensive structural and functional investigation of PAC by revealing its pH-sensing residues and the associated structural mechanisms (Aim 1). I will also explore strategies to obtain an open state structure of PAC and provide detailed mechanistic knowledge about its voltage-dependent gating mechanisms (Aim 2). I will also study the PAC channel in its native state by purifying endogenous PAC protein from mouse brain (Aim 3). Lastly, I will investigate small molecule-mediated inhibition mechanisms through combined structural and functional approaches (Aim 4). The mentored phase of the award will be conducted at Van Andel Institute under the supervision of Dr. Juan Du. During this time, I will receive additional training in membrane protein structure determination, patch-clamp electrophysiology experiments, and endogenous protein purification techniques. These components are not only essential for the completion of the research but will also prepare me to become an independent investigator in the near future.

抽象的 缺血性中风是美国残疾和死亡的主要原因之一。酸积聚于 缺血性中风期间的大脑会导致神经毒性和不可逆的组织损伤。了解 因此，在缺血性中风期间导致酸诱导细胞死亡的因素对于定义缺血性中风期间的酸诱导细胞死亡至关重要。 病理过程并制定有效的治疗策略。质子激活氯离子 (PAC) 通道 （也称为 ASOR 或 PAORAC）是最近发现的一种细胞 pH 传感器，在 确定缺血性中风后脑损伤的结果。在酸性条件下，PAC的活化 允许氯电流流入神经元，进一步导致细胞肿胀和死亡。 2019年， PAC基因由两个独立的小组克隆，并被发现是一种新型的氯离子通道。 2020年，我 揭示了人类 PAC 通道在两种不同构象下的第一个近原子冷冻电镜结构 态，包括apo态和质子束缚非导电态。我们的研究提供了丰富的 有关 PAC 的通道化学计量、域结构和阴离子选择性机制的信息。尽管 在了解该通道的功能方面已经取得了可喜的进展，全面了解了 由于对 pH 值的了解有限，PAC 如何应对环境酸化仍不清楚。 传感器和缺乏开放状态结构。同样，虽然 PAC 电流对几种非敏感 特定的氯离子通道阻滞剂，其抑制机制尚未探索。本次活动的长远目标 研究旨在揭示 PAC 通道功能在生理和心理方面的分子原理 病理条件，并开发可用于减轻影响的特定化合物 缺血性中风患者。在本次K99/R00提案中，将进行全面的结构和功能 通过揭示 PAC 的 pH 感应残基和相关的结构机制来研究 PAC（目标 1）。我 还将探索获得 PAC 开放状态结构的策略，并提供详细的机制 关于其电压依赖性门控机制的知识（目标 2）。我还将研究 PAC 渠道 通过从小鼠大脑中纯化内源性 PAC 蛋白来达到天然状态（目标 3）。最后我会调查一些小事 通过结构和功能相结合的方法实现分子介导的抑制机制（目标 4）。 该奖项的指导阶段将在胡安博士的监督下在范安德尔研究所进行 杜。在此期间，我将接受膜蛋白结构测定、膜片钳等方面的额外培训 电生理学实验和内源性蛋白质纯化技术。这些组件不是 不仅对于完成研究至关重要，而且也让我为成为一名独立的人做好准备 调查员在不久的将来。