Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

Opening of mitochondrial K+ channels increases ischemic ATP levels by preventing hydrolysis

Mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK_ATP in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK_ATP opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca²⁺ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK_ATP-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K⁺ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨ_m). At K⁺ concentrations within the range of 0–120 mM, an increase in the initial rate of K⁺-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K⁺-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K _ATP ⁺ -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K⁺ uptake. The ATP-dependent transport of K⁺ was shown to be ～40% of the potential-dependent K⁺ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K⁺ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K _ATP ⁺ -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.     

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect

In the present study, we describe the existence of mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoK_ATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoK_ATP in insect mitochondria. MitoK_ATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.     

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

Inhibition of oxygen consumption in skeletal muscle-derived mitochondria by pinacidil,diazoxide, and glibenclamide,but not by 5-hydroxydecanoate

Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (K_ATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates and whether these changes are affected by K_ATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and K_ATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when the selective mitochondrial K_ATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these K_ATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-K_ATP or other respiratory-chain components.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K⁺ Channel (PmitoK_ATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K⁺ uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoK_ATP and ⁺/H⁺ antiporter allows such a great operation of a K⁺ cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoK_ATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics. [BMB Reports 2013; 46(8): 391-397]     

Mitochondrial K(ATP) channel openers activate the ERK kinase by an oxidant-dependent mechanism

Extracellularsignal-regulated kinases (ERKs) are key regulatory proteins thatmediate cell survival, proliferation, and differentiation. Reactiveoxygen species (ROS) may play a role in activation of the ERK pathway.Because mitochondria are a major source of ROS, we investigated whethermitochondria-derived ROS play a role in ERK activation. Diazoxide, apotent mitochondrial ATP-sensitive K⁺ (K_ATP)channel opener, is known to depolarize the mitochondrial membranepotential and cause a reversible oxidation of respiratory chainflavoproteins, thus increasing mitochondrial ROS production. UsingTHP-1 cells as a model, we postulated that opening mitochondrial K_ATP channels would increase production of ROS and,thereby, regulate the activity of the ERK kinase. We found that openingmitochondrial K_ATP channels by diazoxide inducedproduction of ROS as determined by an increased rate of dihydroethidiumand dichlorofluorescein fluorescence. This increased production of ROSwas associated with increased phosphorylation of ERK kinase in atime-dependent fashion. The MEK inhibitors PD-98059 and U-0126 blockedERK activation mediated by diazoxide. N-acetylcysteine, butnot diphenyleneiodonium, attenuated ERK activation mediated bydiazoxide. Adenovirus-mediated overexpression of manganese superoxidedismutase, which is expressed in mitochondria, decreased the rate ofdihydroethidium oxidation as well as ERK activation. We conclude thatmitochondrial K_ATP channel openers trigger ERK activationvia mitochondria-derived ROS.

     

MitoK<Subscript>ATP</Subscript> activity in healthy and ischemic hearts

In addition to their role in energy transduction, mitochondria play important non-canonical roles in cell pathophysiology, several of which utilize the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP). In the normal heart, mitoK_ATP regulates energy transfer through its regulation of intermembrane space volume and is accordingly essential for the inotropic response during periods of high workload. In the ischemic heart, mitoK_ATP is the point of convergence of protective signaling pathways and mediates inhibition of the mitochondrial permeability transition, and thus necrosis. In this review, we outline the experimental evidence that support these roles for mitoK_ATP in health and disease, as well as our hypothesis for the mechanism by which complex cardioprotective signals that originate at plasma membrane receptors traverse the cytosol to reach mitochondria and activate mitoK_ATP.     

Tl+ induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria

Effects of Tl⁺ were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO₃ and a nitrate salt (KNO₃ or NH₄NO₃) or TlNO₃ and sucrose. Tl⁺ increased permeability of the inner membrane of the RHM to K⁺ and H⁺. This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K⁺ < NH₄ ⁺, respectively. After succinate administration, the swollen RHM contracted. The Tl⁺-induced opening of the mitochondrial permeability pore (MPTP) in Ca²⁺-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoK_ATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoK_ATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl⁺ effects on muscle cell mitochondria.     

Effect of potential-dependent potassium uptake on calcium accumulation in rat brain mitochondria

The effect of potential-dependent potassium uptake at 0–120 mM K⁺ on matrix Ca²⁺ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K⁺ content in the medium was observed due to K⁺ uptake into the mitochondria. The accumulation of Ca²⁺ was shown to depend on K⁺ concentration in the medium. At K⁺ concentration ?30 mM, Ca²⁺ uptake is decreased due to K⁺-induced membrane depolarization, whereas at higher K⁺ concentrations, up to 120 mM K⁺, Ca²⁺ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K⁺ uptake. Mitochondrial K _ATP ⁺ -channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K⁺ uptake as well as K⁺-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca²⁺ uptake, i.e. increase in Ca²⁺ uptake at low K⁺ content in the medium due to the smaller membrane depolarization and decrease in Ca²⁺ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K _ATP ⁺ channel, in the regulation of calcium accumulation in rat brain mitochondria.     

The Intracellular Localization and Function of the ATP-Sensitive K+ Channel Subunit Kir6.1

Our aim was to determine the subcellular localization and functional roles of the K_ATP channel subunit Kir6.1 in intracellular membranes. Specifically, we focused on the potential role of Kir6.1 as a subunit of the mitochondrial ATP-sensitive K⁺ channel. Cell imaging showed that a major proportion of heterologously expressed Kir6.1-GFP and endogenously expressed Kir6.1 was distributed in the endoplasmic reticulum with little in the mitochondria or plasma membrane. We used pharmacological and molecular tools to investigate the functional significance of this distribution. The K_ATP channel opener diazoxide increased reactive oxygen species production, and glibenclamide abolished this effect. However, in cells lacking Kir6.1 or expressing siRNA or dominant negative constructs of Kir6.1, the same effect was seen. Ca²⁺ handling was examined in the muscle cell line C2C12. Transfection of the dominant negative constructs of Kir6.1 significantly reduced the amplitude and rate of rise of [Ca²⁺] _c transients elicited by ATP. This study suggests that Kir6.1 is located in the endoplasmic reticulum and plays a role in modifying Ca²⁺ release from intracellular stores.     

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)). Regulation of the mitoK(ATP) in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoK(ATP) during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoK(ATP) activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2',7'-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3'-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoK(ATP), depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK(ATP) blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated value for diazoxide to mitoK(ATP) under normoxia was four times higher than under oxidative stress conditions: 5.01 +/- 1.47-10(-6) M and 1.24 +/- 0.21 x 10(-6) M respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoK(ATP) under oxidative stress. Taking into account the higher affinity of diazoxide to mitoK(ATP) under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoK(ATP). Thus we hypothesize that the ROS regulate the activity of the mitoK(ATP) in myocytes.     

Effect of mitochondrial ATP-dependent potassium channel effectors diazoxide and glybenclamide on hydrodynamic diameter and membrane potential of the myometrial mitochondria

Mitochondrial ATP-sensitive potassium channel (mitoKATP) is a main factor of regulation of K+ exchange in mitochondria. Using photon correlation spectroscopy we have shown diazoxide-induced increase of hydrodynamic diameter of mitochondrial particles from the rat myometrium. Selective channel blocker glybenclamide partially eliminates this effect. By means of Rhodamine-123 fluorescence it was shown that activation of ATP-sensitive K(+)-channel in mitochondria caused partial depolarization of the mitochondrial membrane. This effect was absolutely blocked by glybenclamide. In the presence of valinomycine and diazoxide together, depolarization also was detected, but in this case glybenclamide failed to restore mitochondrial potential. Thus, activation of mitoKATP from the rat myometrium causes the increase of the hydrodynamic diameter of organelles and partial depolarization of the inner membrane.     

Interaction of mitochondrial potassium channels with the permeability transition pore

Three types of potassium channels cooperate with the permeability transition pore (PTP) in the inner mitochondrial membranes of various tissues, mtK_(ATP), mtBK, and mtKv1.3. While the latter two share similarities with their plasma membrane counterparts, mtK_(ATP) exhibits considerable differences with the plasma membrane K_(ATP)-channel. One important function seems to be suppression of release of proapototic substances from mitochondria through the PTP. Open potassium channels tend to keep the PTP closed thus acting as antiapoptotic. Nevertheless, in their mode of action there are considerable differences among them. This review introduces three K⁺-channels and the PTP, and discusses known facts about their interaction.     

A role of opening of mitochondrial ATP-sensitive potassium channels in the infarct size-limiting effect of ischemic preconditioning via activation of protein kinase C in the canine heart

The opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels triggers or mediates the infarct size (IS)-limiting effect of ischemic preconditioning (IP). Because ecto-5′-nucleotidase related to IP is activated by PKC, we tested whether the opening of mitoK_ATP channels activates PKC and contributes to either activation of ecto-5′-nucleotidase or IS-limiting effect. In dogs, IP procedure decreased IS and activated ecto-5′-nucleotidase, both of which were mimicked by transient exposure to either cromakalim or diazoxide, and these effects were blunted by either GF109203X (a PKC inhibitor) or 5-hydroxydecanoate (a mitoK_ATP channel blocker), but not by HMR-1098 (a surface sarcolenmal K_ATP channel blocker). Either cromakalim or diazoxide activated both PKC and ecto-5′-nucleotidase, which was blunted by either GF109203X or 5-hydroxydecanoate, but not by HMR-1098. We concluded that the opening of mitoK_ATP channels contributes to either activation of ecto-5′-nucleotidase or the infarct size-limiting effect via activation of PKC in canine hearts.     

Glutamine Transport in Rat Brain Synaptic and Non-Synaptic Mitochondria

Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [³H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K⁺/H⁺ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K⁺, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher K_m value. This uptake is inhibited by valinomycin in the presence of K⁺ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.