The LRRC8 volume\regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel
The LRRC8 volume\regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel. mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the Rabbit Polyclonal to PTPRN2 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. was used; for mitochondrial membrane potential a one\way ANOVA was used with post hoc Tukey’s test. Significance is usually indicated as: *(current density at ?120?mV from WT and LRRC8A\KO HAP1 cells measured using a ramp protocol from ?120?mV and +120?mV under the indicated bath solution conditions ((((TMRE fluorescence of (e) WT or (f) LRRC8A\KO cells measured with flow cytometry. SB-423562 Statistical analysis was carried out using one\way ANOVA and Tukey’s post hoc analysis. *indicates (oocytes (Deng et al., 2016). Inhibition was also observed in a Kir6.2 C\terminal truncation mutant that allows the channel to be expressed in the absence of SUR1, indicating that the DCPIB binding site is located in the pore\forming Kir6.2 subunit. Taken together with our data, SB-423562 the apparently poor activation of Kir6.2/SUR1 observed in the present study might reflect a combination of Kir6.2/SUR1 activation and inhibition by DCPIB. It is important to note that in cells lacking LRRC8A expression, the remaining LRRC8B\E subunit are still expressed (Voss et al., 2014). It is conceivable, albeit unlikely given what is currently known about the requirement of LRRC8A for the assembly of functional VRAC channels (Qiu et al., 2014; Voss et al., 2014), that other LRRC8 subunits form DCPIB receptors in mitochondria. It will be important in future studies to determine if different LRRC8 subunits are expressed in mitochondria and contribute to respiration. In conclusion, we have shown that the current best\in\class inhibitor of VRAC, DCPIB, suppresses mitochondrial respiration and ATP production by uncoupling the mitochondrial proton gradient and inhibiting complexes I, II, and III of the ETC. Because these effects are observed in cells lacking the expression of the essential VRAC subunit, LRRC8A, they are likely unrelated to inhibition of VRAC channel function. This study emphasizes the need for improved pharmacological tools to investigate the integrative physiology of the channel where metabolism could be an important factor. We recently reported the discovery of two CysLT1 receptor antagonists, Pranlukast and Zafirlukast, as novel\scaffold inhibitors of VRAC (Figueroa, Kramer, Strange, & Denton, 2019). These compounds could be used as starting points in lead optimization with medicinal chemistry to develop analogs with improved potency and specificity for VRAC. In light of the data presented here, it will be important to evaluate lead compounds for activity toward mitochondrial respiration. AUTHOR CONTRIBUTIONS AA, EF, SVK, JSD, BKM, DKF?and KB contributed to research design. AA, EF, BKM, DKF?and SVK conducted experiments. AA, SB-423562 EF, and SVK performed data analysis. AA, EF, SVK, and JSD wrote manuscript. ACKNOWLEDGMENTS This work was supported by National Institute of Diabetes and Digestive SB-423562 Kidney Disease grants R01 DK51610 (J. Denton) and 1F31DK120225\01 (E. Figueroa), and institutional funds (to A. Afzal and J. Denton). Notes Afzal A, Figueroa EE, Kharade SV, et al. The LRRC8 volume\regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel. Physiol Rep. 2019;7:e14303 10.14814/phy2.14303.