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

摘要 缺血性中风是美国残疾和死亡的主要原因之一。酸积累 缺血性中风期间的脑引起神经毒性和不可逆的组织损伤。了解 因此，在缺血性卒中期间，有助于酸诱导细胞死亡的因素对于确定 病理过程，并制定有效的治疗策略。质子激活氯离子（PAC）通道 (also称为ASOR或PAORAC）是最近发现的细胞pH传感器， 确定缺血性中风后脑损伤的结果。在酸性条件下， 允许氯电流流入神经元，这进一步导致细胞肿胀和死亡。2019年 PAC基因是由两个独立的小组克隆的，并被发现是一个新的氯离子通道。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-EM结构， 状态，包括APO状态和质子束缚的非导电状态。我们的研究提供了丰富的 信息渠道化学计量，域架构，阴离子选择性机制的PAC。而 在了解这一通道的功能方面已经取得了可喜的进展， PAC如何响应环境酸化仍然是模糊的，由于有关pH值的知识有限， 传感器和缺乏开放状态结构。同样，虽然PAC电流对几个非- 特异性氯离子通道阻断剂，其抑制机制尚未探索。长期目标是 这项研究的目的是揭示PAC通道在生理和生理上功能的分子原理， 病理条件，并开发特定的化合物，可用于减轻的影响， 缺血性中风患者。在这个K99/R 00的建议，将进行全面的结构和功能 通过揭示PAC的pH敏感残基和相关的结构机制来研究PAC（目的1）。我 我还将探讨获得PAC开放状态结构的策略，并提供详细的机制， 了解其电压依赖性门控机制（目标2）。我还将研究PAC通道， 通过从小鼠脑中纯化内源性PAC蛋白质来获得天然状态（Aim 3）。最后，我将研究小 分子介导的抑制机制，通过结合结构和功能的方法（目的4）。 该奖项的指导阶段将在Juan博士的监督下在货车Andel研究所进行 杜在此期间，我将接受膜蛋白结构测定、膜片钳 电生理学实验和内源性蛋白质纯化技术。这些组件没有 这对完成研究至关重要，但也将使我成为一个独立的人。 在不久的将来调查。

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.