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