Previous studies showed a poor correlation between sarcolemmal K+ currents and cardioprotection for ATP-sensitive K+ channel (KATP) openers. Diazoxide is a weak cardiac sarcolemmal KATP opener, but it is a potent opener of mitochondrial KATP, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart KATP, diazoxide opened mitochondrial KATP with a K1/2 of 0.8 mumol/L while being 1000-fold less potent at opening sarcolemmal KATP. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC25, 11.0 and 8.8 mumol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. While-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K+ currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac KATP activity. Cromakalim and diazoxide were both potent activators of K+ flux in this preparation (K1/2 values, 1.1 +/- 0.1 and 0.49 +/- 0.05 mumol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K+ flux through the diazoxide-opened mitochondrial KATP. The profile of activity of diazoxide (and perhaps KATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial KATP.

Potassium transport plays three distinct roles in mitochondria. Volume homeostasis to prevent excess matrix swelling is a housekeeping function that is essential for maintaining the structural integrity of the organelle. This function is mediated by the K(+)/H(+) antiporter and was first proposed by Peter Mitchell. Volume homeostasis to prevent excess matrix contraction is a recently discovered function that maintains a fully expanded matrix when diffusive K(+) influx declines due to membrane depolarization caused by high rates of electron transport. Maintaining matrix volume under these conditions is important because matrix contraction inhibits electron transport and also perturbs the structure-function of the intermembrane space (IMS). This volume regulation is mediated by the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Cell signaling functions to protect the cell from ischemia-reperfusion injury and also to trigger transcription of genes required for cell growth. This function depends on the ability of mitoK(ATP) opening to trigger increased mitochondrial production of reactive oxygen species (ROS). This review discusses the properties of the mitochondrial K(+) cycle that help to understand the basis of these diverse effects.

Plasma membrane K ATP channels are highly sensitive to the family of drugs known as K ؉ channel openers, raising the question whether mitochondrial K ATP channels are similarly sensitive to these agents. We addressed this question by measuring K ؉ flux in intact rat liver mitochondria and in liposomes containing K ATP channels purified from rat liver and beef heart mitochondria. K ؉ channel openers completely reversed ATP inhibition of K ؉ flux in both systems. In liposomes, ATP-inhibited K ؉ flux was restored by diazoxide (K1 ⁄2 ‫؍‬ 0.4 M), cromakalim (K1 ⁄2 ‫؍‬ 1 M), and two developmental cromakalim analogues, EMD60480 and EMD57970 (K1 ⁄2 ‫؍‬ 6 nM). Similar K1 ⁄2 values were observed in intact mitochondria. These potencies are well within the range observed with plasma membrane K ATP channels. We also compared the potencies of these K ؉ channel openers on the plasma membrane K ATP channel purified from beef heart myocytes. The K ATP channel from cardiac mitochondria is 2000-fold more sensitive to diazoxide than the channel from cardiac sarcolemma, indicating that two distinct receptor subtypes coexist within the myocyte. We suggest that the mitochondrial K ATP channel is an important intracellular receptor that should be taken into account in considering the pharmacology of K ؉ channel openers.K ϩ channel openers (KCOs) 1 activate ATP-inhibited K ATP channels. As described in several excellent reviews (1-3), members of this drug family exhibit a rich and clinically important pharmacology. Thus, cell membrane K ATP channels (cellK ATP ) in different tissues are considered to mediate the hypotensive and diabetogenic effects of diazoxide (4) and the cardioprotective effects of cromakalim and its derivatives (5). It is important to determine whether these drugs also act on mitochondrial K ATP channels (mitoK ATP ) in their therapeutic range.In the first reports of KCO actions in mitochondria, Belyaeva et al. (6) and Szewczyk et al. (7) observed stimulation of K ϩ uptake by KCOs in respiring mitochondria. RP66471 was the most potent KCO studied (K1 ⁄2 ϭ 50 M), whereas P1060 and diazoxide were only weakly active at 700 M. Because these concentrations are much higher than K1 ⁄2 values observed with cellK ATP (1), these results appear to imply that mitochondrial actions of KCOs are not pharmacologically important.We now report that diazoxide, cromakalim, and two experimental benzopyran derivatives are very potent activators of K ϩ flux through ATP-inhibited mitoK ATP , with K1 ⁄2 values similar to those observed with cellK ATP . KCO activation of K ϩ flux was observed in both intact mitochondria and proteoliposomes containing reconstituted mitoK ATP . No effect was observed on uninhibited K ϩ flux, which likely explains the low potencies observed by previous workers (6, 7) in assays that did not include Mg 2ϩ and ATP. We also found that mitoK ATP and cellK ATP from beef heart differed strongly in their sensitivity to diazoxide, indicating distinct receptor subtypes among K ATP channels from the same cell. Our result...

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.

Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to measure H (ii) Lauric acid was rapidly transported across the bilayer by nonionic diffusion, whereas undecanesulfonic was not. We infer that the role of uncoupling protein in H ؉ transport is to transport fatty acid anions and that fatty acids induce H ؉ transport because they can diffuse electroneutrally across the membrane. According to this hypothesis, uncoupling protein is a pure anion porter and does not transport protons; rather it is designed to enable fatty acids to behave as cycling protonophores.