# Weak Acids as Endogenous Inhibitors of the Proton-Activated Chloride Channel

## TL;DR

This study shows that weak acids like acetic and lactic acid inhibit a proton-activated chloride channel through a specific binding site involving Arg93.

## Contribution

The paper identifies a novel extracellular mechanism by which weak acids inhibit the PAC channel, involving a specific amino acid residue.

## Key findings

- Weak acids inhibit PAC channels in a reversible, concentration-dependent, and pH-dependent manner.
- Extracellular arginine 93 is crucial for the inhibition of PAC channels by certain weak acids.
- Lactic acid inhibits PAC channels but does not reduce proton-induced cytotoxicity in HEK293 cells.

## Abstract

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed, and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites, here, we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells, PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs, and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible, concentration-dependent, and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid, propionic acid, itaconic acid, and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids, including arachidonic acid. Although lactic acid inhibited PAC, it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude, weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids.