Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

Abstract

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O2 concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH2-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O2•−); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H2O2 production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca2+-sensitive K+ channel (mtBKCa) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2 H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O2 consumption, membrane potential (ΔΨm), H2O2 release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 μM) increased state 2 and state 4 respiration by 26 ± 4% and 14 ± 4%, respectively; this increase was abolished by the BKCa channel blocker paxilline (5 μM). Paxilline alone had no effect on respiration. NS-1619 did not alter ΔΨm or redox state but decreased H2O2 production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K+ influx through mtBKCa channels reduces mitochondrial H2O2 production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H2O2 production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K+ influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.