Molecular determinants underlying volume‐regulated anion channel subunit‐dependent oxidation sensitivity

ABSTRACT: The volume‐regulated anion channel (VRAC) is formed by LRRC8 subunits. Besides their role in the maintenance of cell homeostasis, VRACs are critically involved in oxidative stress mechanisms: reactive oxygen species directly modulate VRACs in a subunit‐dependent manner. It was reported that LRRC8A–LRRC8E heteromeric channels are activated by oxidation, whereas LRRC8A–LRRC8C heteromers are inhibited. Here we adopted chimeric‐ as well as concatemeric‐based strategies to identify residues responsible for the divergent effect of oxidants. We identified two cysteines in the first two leucine rich repeats of LRRC8E, C424 and C448, as the targets of oxidation. Oxidation likely results in the formation of a disulfide bond between the two cysteines, which in turn induces a conformational change leading to channel activation. Additionally, we found that LRRC8C inhibition is caused by oxidation of the first methionine. We thus identified crucial molecular elements involved in channel activation, which are conceivably relevant in determining physiological ROS effects. [Image: see text] KEY POINTS: Volume‐regulated anion channels (VRACs) are heterohexameric complexes composed of an essential LRRC8A subunit and a variable number of LRRC8B–E subunits. VRACs are directly regulated by oxidation, with LRRC8A–LRRC8E heteromers being potentiated and LRRC8A–LRRC8C heteromers being inhibited by oxidation. We identified two LRRC8E specific intracellular cysteines that form a disulfide bond upon oxidation leading to LRRC8A–LRRC8E potentiation. Inhibition of LRRC8A–LRRC8C heteromers is mediated by the oxidation of the start methionine, being additionally dependent on the identity of the LRR domain. Besides providing physiological insights concerning the outcome of reactive oxygen species modulation, the results point to key structural elements involved in VRAC activation.