Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca2+-sensitive K+ (mtK(Ca)) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtKCa channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K+ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (Delta Psi(m)). To test this hypothesis, we measured effects of NS-1619, a putative mtK(Ca) channel opener, and valinomycin, a K+ ionophore, on mitochondrial respiration, Delta Psi(m), and ROS generation in guinea pig heart mitochondria. NS-1619 (30 mu M) increased state 2 and 4 respiration by 5.2 +/- 0.9 and 7.3 +/- 0.9 nmol O-2 center dot min(-1)center dot mg protein(-1), respectively, with the NADH-linked substrate pyruvate and by 7.5 +/- 1.4 and 11.6 +/- 2.9 nmol O-2 center dot min(-1)center dot mg protein(-1), respectively, with the FADH(2)-linked substrate succinate (+ rotenone); these effects were abolished by the mtK(Ca) channel blocker paxilline. Delta Psi(m) was not decreased by 10-30 mu M NS-1619 with either substrate, but H2O2 release was increased by 44.8% (65.9 +/- 2.7% by 30 mu M NS-1619 vs. 21.1 +/- 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H2O2 release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained Delta Psi(m), despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K+ influx during states 2 and 4 increases mitochondrial respiration while maintaining Delta Psi(m); this allows singlet electron uptake by O-2 and ROS generation.