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

Abstract

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca(2+)-sensitive K(+) (mtK(Ca)) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK(Ca) 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 (DeltaPsi(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, DeltaPsi(m), and ROS generation in guinea pig heart mitochondria. NS-1619 (30 microM) increased state 2 and 4 respiration by 5.2 +/- 0.9 and 7.3 +/- 0.9 nmol O(2).min(-1).mg protein(-1), respectively, with the NADH-linked substrate pyruvate and by 7.5 +/- 1.4 and 11.6 +/- 2.9 nmol O(2).min(-1).mg protein(-1), respectively, with the FADH(2)-linked substrate succinate (+ rotenone); these effects were abolished by the mtK(Ca) channel blocker paxilline. DeltaPsi(m) was not decreased by 10-30 microM NS-1619 with either substrate, but H(2)O(2) release was increased by 44.8% (65.9 +/- 2.7% by 30 muM NS-1619 vs. 21.1 +/- 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H(2)O(2) 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 DeltaPsi(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 DeltaPsi(m); this allows singlet electron uptake by O(2) and ROS generation.