The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

There has been a resurgence of interest in the volume‐regulated anion channel (VRAC) since the recent cloning of the LRRC8A‐E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best‐in‐class VRAC inhibitor, DCPIB, suffers from off‐target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A‐knockout HAP‐1 and HEK‐293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP‐1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP‐linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP‐1 and HEK‐293 cells which lack LRRC8A expression. Finally, we demonstrate that DCPIB activates ATP‐inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I‐III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases.