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

摘要 在美国，缺血性中风是导致残疾和死亡的主要原因之一。酸在土壤中积累 缺血性中风时的大脑会引起神经毒性和不可逆的组织损伤。了解 因此，在缺血性卒中中，导致酸诱导的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。质子活化氯(PAC)通道 (也称为ASOR或PAORAC)是最近发现的一种细胞pH传感器，在 确定缺血性卒中后脑损伤的转归。在酸性条件下，PAC的活化 允许氯电流流入神经元，从而进一步导致细胞肿胀和死亡。2019年， PAC基因是由两个独立的群体克隆的，被发现是一种新的氯通道。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低温EM结构 态，包括脱氧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、结构域结构和阴离子选择性机制的信息。而当 在理解这个渠道的功能方面取得了可喜的进展，全面了解了 由于对pH值的了解有限，PAC如何响应环境酸化仍然是个未知数。 传感器和缺乏开放的状态结构。同样，尽管PAC电流对几个非 具体的氯通道阻滞剂，其抑制机制尚不清楚。这样做的长期目标是 研究旨在揭示PAC通道在生理和生理功能方面的分子机制 病理条件，并开发特定的化合物，可以用来减轻 缺血性卒中患者。在这份K99/R00提案中，将进行全面的结构和功能 通过揭示其对pH敏感的残基和相关的结构机制来研究PAC(目标1)。我 还将探索策略，以获得PAC的开放状态结构并提供详细的机制 关于其电压依赖门控机制的知识(目标2)。我亦会研究政府账目委员会在其 通过从小鼠脑中纯化内源性PAC蛋白实现自然状态(目标3)。最后，我会调查Small 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范安德尔研究所进行，由胡安博士监督 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 不仅对完成研究是必不可少的，而且还将使我准备成为一个独立的 在不久的将来成为一名调查员。

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.