Determination of the rate of K+ movement through potassium channels in isolated rat heart and liver mitochondria

Both ATP-regulated (mitoKATP) and large conductance calcium-activated (mitoBKCa) potassium channels have been proposed to regulate mitochondrial K+ influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K+-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K+/H+ exchanger nigericin. This allowed rates of valinomycin-induced K+ influx to be determined whilst parallel measurements of light scattering (A520) and matrix volume (3H2O and [14C]-sucrose) enabled rates of K+ influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K+ influx of < 5 nmol K+ min− 1 per mg protein corresponding to < 2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoKATP channel openers or blockers. However, the mitoBKCa opener NS1619 (10–50 µM) did decrease light scattering slightly, but this was also seen in K+-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBKCa blocker paxilline (10–50 µM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoKATP or the mitoBKCa channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.