Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism
LRRC8 volume-regulated anion channels (VRACs) play important roles in diverse cell types and may represent therapeutic targets for diseases. To date, however, the pharmacological tools for evaluating the druggability of VRAC have been limited to inhibitors, as no activators of the channel have been reported. We performed a fluorescence-based high-throughput screen (HTS) of 1,184 FDA-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially for treating dandruff and other skin disorders. In intracellular YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose-dependently with a half-maximal effective concentration (EC50) of 5.7 µM. Whole-cell voltage-clamp experiments revealed that co-application of hypotonic solution and 30 µM ZPT to HEK293 or HCT116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC, however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger NAC and NAD(P)H oxidase inhibitor DPI, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.