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.

Coronary artery disease and its sequelae-ischemia, myocardial infarction, and heart failure-are leading causes of morbidity and mortality in man. Considerable effort has been devoted toward improving functional recovery and reducing the extent of infarction after ischemic episodes. As a step in this direction, it was found that the heart was significantly protected against ischemia-reperfusion injury if it was first preconditioned by brief ischemia or by administering a potassium channel opener. Both of these preconditioning strategies were found to require opening of a K(ATP) channel, and in 1997 we showed that this pivotal role was mediated by the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). This paper will review the evidence showing that opening mitoK(ATP) is cardioprotective against ischemia-reperfusion injury and, moreover, that mitoK(ATP) plays this role during all three phases of the natural history of ischemia-reperfusion injury preconditioning, ischemia, and reperfusion. We discuss two distinct mechanisms by which mitoK(ATP) opening protects the heart-increased mitochondrial production of reactive oxygen species (ROS) during the preconditioning phase and regulation of intermembrane space (IMS) volume during the ischemic and reperfusion phases. It is likely that cardioprotection by ischemic preconditioning (IPC) and K(ATP) channel openers (KCOs) arises from utilization of normal physiological processes. Accordingly, we summarize the results of new studies that focus on the role of mitoK(ATP) in normal cardiomyocyte physiology. Here, we observe the same two mechanisms at work. In low-energy states, mitoK(ATP) opening triggers increased mitochondrial ROS production, thereby amplifying a cell signaling pathway leading to gene transcription and cell growth. In high-energy states, mitoK(ATP) opening prevents the matrix contraction that would otherwise occur during high rates of electron transport. MitoK(ATP)-mediated volume regulation, in turn, prevents disruption of the structure-function of the IMS and facilitates efficient energy transfers between mitochondria and myofibrillar ATPases.

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K ATP channel openers is known to involve the mitochondrial ATPsensitive K ؉ channel (mitoK ATP ). Brain is also protected by ischemic preconditioning and K ATP channel openers, and it has been suggested that mitoK ATP may also play a key role in brain protection. However, it is not known whether mitoK ATP exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK ATP . We report partial purification and reconstitution of a new mitoK ATP from rat brain mitochondria. We measured K ؉ flux in proteoliposomes and found that brain mitoK ATP is regulated by the same ligands as those that regulate mitoK ATP from heart and liver. We also examined the effects of opening and closing mitoK ATP on brain mitochondrial respiration, and we estimated the amount of mitoK ATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK ATP from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK ATP per milligram of mitochondrial protein than liver or heart.

. Mechanisms by which opening the mitochondrial ATP-sensitive K ϩ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol 283: H284-H295, 2002. First published February 14, 2002 10.1152/ajpheart. 00034.2002-Diazoxide opening of the mitochondrial ATPsensitive K ϩ (mitoKATP) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoKATP channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoKATP channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK ATP channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion. mitochondria; metabolism; creatine kinase; membrane transport; cytochrome c; ischemic preconditioning THERE IS NOW GENERAL AGREEMENT that the mitochondrial ATP-sensitive K ϩ (mitoK ATP ) channel plays a pivotal role in cardioprotection against ischemia-reperfusion injury (16,17,20,21,36,64); however, little is known about the mechanism of this protection. It has been proposed that mitoK ATP channel opening triggers protection by increasing the generation of reactive oxygen species (ROS) (13, 47), and this effect has now been demonstrated by several laboratories (10,13,44,57). We proposed that the mitoK ATP channel is also an end effector of protection (16), and many studies have confirmed that the mitoK ATP channel is required to be open during the ischemic phase (11,46,58,59,64). Thus the mitoK ATP channel is both a trigger and an end effector of cardioprotection, and these two roles are temporally and mechanistically distinct.We have previously suggested that cardioprotection by mitoK ATP channel opening is due in part to volume regulation, which serves to preserve the structurefunction of the intermembrane space (IMS) and the low permeability of the outer membrane to nucleotides (31). Nucleotide transport across the outer membrane occurs primarily through the voltage-dependent anion channel (VDAC) (2, 34, 49, 50). We hypothesize that VDAC permeability to nucleotides is regulated in part by IMS volume, which in turn is regulated by K ϩ flux across the inner membrane.This study focuses on the end effector mechanisms by which an open mitoK ATP channel protects the heart during ischemia and reperfusion. We show, in sapon...

The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK ATP ) is a highly selective conductor of K ؉ ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoK ATP is inhibited with high affinity by ATP (K 1/2 ‫؍‬ 20 -30 M). We have suggested that holo-mitoK ATP is a heteromultimer consisting of an inwardly rectifying K The importance of the mitochondrial K ϩ cycle for volume regulation, reviewed by Garlid and Paucek (1) was recognized by Mitchell (2) long before any of the components of this cycle were discovered. K ϩ is driven into the matrix by the high membrane potential (⌬⌿) 1 generated by the proton-pumping electron transport system, and excess K ϩ is removed by the regulated K ϩ /H ϩ antiporter. Electrophoretic K ϩ influx occurs by diffusion and by means of ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK ATP ). At the high values of ⌬⌿ maintained by mitochondria, both of these processes increase exponentially with ⌬⌿ (3, 4) and are consequently very sensitive to fluctuations in ⌬⌿. These fluctuations, in turn, are high in tissues such as heart, which undergo large variations in energy demand and ATP synthesis rates (5). Thus, regulation of K ϩ influx and efflux pathways can be seen as a means of regulating volume in the face of the changing energy requirements of the cell. MitoK ATP plays more than a housekeeping role in cell physiology. There is now general agreement that mitoK ATP plays a key role in cardioprotection against ischemia-reperfusion injury (6, 7). The proposed mechanisms of this protective effect of mitoK ATP opening (5, 8, 9) are plausible; however, it is evident that more needs to be known about the functional properties of mitoK ATP before its role in vivo can be ascertained.By using a novel ethanol extraction technique, Mironova et al. (10) were the first to report reconstitution in lipid bilayer membranes of a 55-kDa K ϩ channel from mitochondria. Paucek et al. (3) used a detergent extraction technique and were the first to report reconstitution of mitoK ATP in liposomes. The latter channel was associated with two proteins of molecular mass 55 and 63 kDa, and we hypothesized that mitoK ATP is a heteromultimeric complex consisting of a 55-kDa inwardly rectifying K ϩ channel (mitoKIR) and a 63-kDa sulfonylurea receptor (mitoSUR), analogous to the plasma membrane ATPdependent K ϩ channel (cellK ATP ) (11,12). In this report, we focus on three interactions that address the key question of whether the 55-kDa K ϩ channel protein observed in the ethanol purification is the same as the 55-kDa protein purified with detergents. First, we show that UDP reverses ATP-inhibition of K ϩ flux mediated by both mitoKIR and mitoK ATP reconstituted in liposomes. Moreover, UDP exerts the same action in isolated mitochondria, and the affinities for the opening effect of UDP are about the same in each preparation. Second, we show that the mitoKIR opener p-diethylaminoethylbenzoate (DEB) also activates K ϩ flux via ...