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

> **NIH NIH R00** · THOMAS JEFFERSON UNIVERSITY · 2024 · $241,529

## Abstract

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.

## Key facts

- **NIH application ID:** 10973763
- **Project number:** 4R00NS128258-03
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Zheng Ruan
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $241,529
- **Award type:** 4N
- **Project period:** 2022-08-15 → 2027-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10973763

## Citation

> US National Institutes of Health, RePORTER application 10973763, Structural and Functional Studies on Proton-activated Chloride (PAC) Channel (4R00NS128258-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10973763. Licensed CC0.

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