Mitochondrial ATP-regulated Potassium Channel Research Articles

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Influence of Mitochondrial ATP-Sensitive Potassium Channels on Toxic Effect of Amyloid-β 25–35

Amyloid-β (Aβ) is the main component of senile plaques, one of the hallmarks of Alzheimer,s disease. Is been shown that Aβ25–35 decreased neuronal viability while it increased generation of reactive oxygen species (ROS), and albumin (BSA) prevented ROS production and neuronal death in a dose-and time-dependent manner. One of the major sources of ROS is mitochondrion, and is believed that Mitochondrial ATP-regulated potassium channels (mitoKATP) protect synapses and neurons against oxidative and metabolic stress by modulating inner membrane potential and ROS production. Here we investigate the possible participation of MitoKATP channels on toxic effect of Aβ and the protective effect of BSA, by studying the influence of diazoxide (DIAZ) and tolbutamide (TOLB) on the effect of Aβ25–35 in neuronal morphology, cell viability and ROS generation in presence and absence of BSA. DIAZ decreased ROS generation induced by Aβ25–35 in a concentration dependent manner, but increased with the addition of BSA. TOLB increased Aβ25–35 effect on ROS production in a concentration dependent manner, but only in presence of BSA. Neither DIAZ nor TOLB rescued neurons from morphological damage and cell death induced by Aβ25–35. Hence, it could be proposed that MitoKATP channels participate on toxic effects of Aβ25–35, but not in protective effect of BSA, which seems to go through an extraneuronal mechanism.

Evidence for a mitochondrial ATP-regulated potassium channel in human dermal fibroblasts

Mitochondrial ATP-regulated potassium channels are present in the inner membrane of the mitochondria of various cells. In the present study, we show for the first time mitochondrial ATP-regulated potassium channels in human dermal fibroblast cells. Using the patch-clamp technique on the inner mitochondrial membrane of fibroblasts, we detected a potassium channel with a mean conductance equal to 100 pS in symmetric 150 mM KCl. The activity of this channel was inhibited by a complex of ATP/Mg2+ and activated by potassium channel openers such as diazoxide or BMS 191095. Channel activity was inhibited by antidiabetic sulfonylurea glibenclamide and 5-hydroxydecanoic acid. The influence of substances modulating ATP-regulated potassium channel activity on oxygen consumption and membrane potential of isolated fibroblast mitochondria was also studied. Additionally, the potassium channel opener diazoxide lowered the amount of superoxide formed in isolated fibroblast mitochondria. Using reverse transcriptase-PCR, we found an mRNA transcript for the KCNJ1(ROMK) channel. The presence of ROMK protein was observed in the inner mitochondrial membrane fraction. Moreover, colocalization of the ROMK protein and a mitochondrial marker in the mitochondria of fibroblast cells was shown by immunofluorescence. In summary, the ATP-regulated mitochondrial potassium channel in a dermal fibroblast cell line have been identified.

Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures

Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

The Cytoprotective Action of the Potassium Channel Opener BMS-191095 in C2C12 Myoblasts is Related to the Modulation of Calcium Homeostasis

BMS-191095 is an opener of the mitochondrial ATP-regulated potassium channel, which has been shown to provide cytoprotection in models of ischemia-reperfusion induced injury in various tissues. This study aimed at checking the protective action of BMS-191095 under the conditions of oxidative stress or disruption of intracellular calcium homeostasis. Methods: The cytoprotective potential of BMS-191095 was tested in C2C12 myoblasts injured by treatment with H₂O₂ or calcium ionophore A23187. The influence of the opener on intracellular calcium levels, calpain activity and respiration rates were determined. Results: BMS-191095 protected myoblasts from calcium ionophore A23187-induced injury, but not from H H₂O₂-induced injury. A23187-mediated cell damage was also prevented by calpain inhibitor PD 150606. A23187 administration led to a transient increase in cytosolic calcium levels, concomitant activation of calpains and a decrease in state 3 respiration rates, indicating mitochondrial dysfunction. Co-administration of BMS-191095 diminished calpain activation in A23187-treated cells but did not prevent mitochondrial damage. In the presence of BMS-191095, restoration of cytosolic calcium concentrations to basal levels after A23187 treatment was considerably faster which may underly the reduced activation of calpains. Conclusion: The BMS-191095-mediated cytoprotection observed in C2C12 myoblasts results probably from modulation of intracellular calcium transients leading to prevention of calpain activation.

Single channel studies of the ATP-regulated potassium channel in brain mitochondria

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 +/- 15 pS. The activity of this channel was inhibited by ATP/Mg(2+) and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.

Large Conductance Potassium Channel In Mitochondria of Endothelial Cell

It is well established that endothelial dysfunction contributes to ischemia-reperfusion injury of cardiovascular system. This phenomenon can be limited by the ischemic preconditioning. Recently, it was shown that mitochondrial ATP regulated potassium channel activation induced ischemic preconditiong of the endothelium in humans in vivo.In our study a single channel activity was measured after patch-clamp of the mitoplasts isolated from endothelial cell line (EA.hy926). Mitoplast samples were prepared by addition to a hypotonic solution causing the cristae of the inner membrane to unfold and breaking of the outer membrane. Isotonicity was restored by adding of hypertonic solution. A potassium selective current was recorded with mean conductance 270 ± 10 pS in symmetrical 150 mM KCl solution. Patch-clamp single channel studies showed properties of the large conductance Ca2+−regulated potassium channel (BKCa channel): it was activated by calcium and NS1619 an activator of BKCa channel at micromolar concentration range. These effects were blocked irreversible by iberiotoxin (IbTx), an inhibitor of BKCa channel. Additionally, we showed that inhibitor of mitoKATP channel (ATP/Mg2+ complex) have no effects on observed activity of ion channel.We conclude that large conductance Ca2+−regulated potassium channels are present in mitochondria isolated from endothelial cell line.This work was supported by Polish Mitochondrial Network MitoNet.pl and grant N30105331/1676

Voltage-gated Potassium Channel In Brain Mitochondria

Transient cerebral ischemia is known to induce endogenous adaptive mechanisms such as the activation of mitochondrial ATP regulated potassium channels or Ca2+ regulated large conductance potassium channels that can prevent or delay neuronal injury. In this study a single channel activity was measured after patch-clamp of the mitoplasts isolated from gerbil hippocampus. In 70% of the all patches, a potassium selective current was recorded with mean conductance 109 ± 6 pS in symmetrical 150 mM KCl solution. The patch-clamp single channel studies showed properties of the voltage-gated potassium channel (Kv channel). We found that ATP/Mg2+ complex and Ca2+ ions had no effects on observed activity of ion channel. Observed channel was blocked by negative voltage and margatoxin (MgTx) a specific Kv1.3 channel inhibitor. The inhibition by MgTx was irreversible. We conclude that gerbil hippocampus mitochondria contain voltage-gated potassium channel (mitoKv) with properties similar to the surface membrane Kv1.3 channel which can play a role in control function of mitochondria as well as in ischemia-reperfusion phenomenon.This study was supported by grant No. P-N/031/2006 and Polish Mitochondrial Network MitoNet.pl.

Cytoprotective Effects Of Mitochondrial Potassium Channel Opener BMS-191095

Mitochondrial potassium channel openers (KCO's) were shown to be cytoprotective in models of ischemia-reperfusion induced injury in brain, heart and skeletal muscle tissue. The aim of this study was to identify the cellular events responsible for observed protection. We have investigated the cytoprotective potential of BMS-191095, an opener of the mitochondrial ATP-regulated potassium channel (mitoKATP), in C2C12 myoblasts. BMS-191095 did not protect the cells against tert-butyl hydroperoxide or H2O2-induced injury, but prevented calcium ionophore A23187-induced cell death. A23187 caused a transient increase in cytosolic calcium levels, which was not affected by the presence BMS-191095. In contrary, the opener prevented a mild elevation of calcium levels observed after the initial peak. This study was supported by Polish Mitochondrial Network MitoNet.pl and Polish Ministry of Science and Higher Education grants no. N30105331/1617 and P-N/031/2006.

New properties of mitochondrial ATP-regulated potassium channels

The ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. In this study, the activity of a single channel was measured after reconstituting the myocardium inner mitochondrial membrane into a planar lipid bilayer. We provide direct evidence of vectorial pH regulation of mitoK(ATP) channels. When the matrix side was alkalized, this changed the channel conductance, the open probability, and the mean open and closed dwell time distributions. The conductance of the mitoK(ATP) channel increased from about 110 +/- 8 to 145 +/- 5 pS upon changing the pH from 7.2 to 8.2. This effect was reversed by reverting the pH to the neutral value. The mitoK(ATP) channel activity was not altered by alkalization of the cytosolic side of the planar lipid bilayer. We also observed that acidification from pH 7.2 to 6.2, in either the matrix or cytosolic compartments, decreased the open probability of the channel. This effect was reversed by perfusion with a pH 7.2 medium. Additionally, our results suggest that the mitoK(ATP) channel is regulated by multiple phosphorylation events. The channel activity was inhibited by an ATP/Mg(2+) complex, but not by ATP alone, nor by a non-hydrolysable ATP analog, e.g. AMP-PNP/Mg(2+). The mitoK(ATP) channel "run-down" was reversed by incubating with the ATP/Mg(2+) complex on both sides of the planar lipid bilayer. We conclude that both pH and ATP play an important regulatory role for the cardiac mitoK(ATP) channel with respect to the phenomenon of ischemia-reperfusion.

Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart.

The mitochondrial ATP-regulated potassium (mitoK(ATP) channel has been suggested as trigger and effector in myocardial ischemic preconditioning. However, molecular and pharmacological properties of the mitoK(ATP) channel remain unclear. In the present study, single-channel activity was measured after reconstitution of the inner mitochondrial membrane from bovine ventricular myocardium into bilayer lipid membrane. After incorporation, a potassium-selective current was recorded with mean conductance of 103 +/- 9 pS in symmetrical 150 mM KCl. Single-channel activity of this reconstituted protein showed properties of the mitoK(ATP) channel: it was blocked by 500 microM ATP/Mg, activated by the potassium-channel opener diazoxide at 30 microM, inhibited by 50 microM glibenclamide or 150 microM 5-hydroxydecanoic acid, and was not affected by the plasma membrane ATP-regulated potassium-channel blocker HMR1098 at 100 microM. We observed that the mitoK(ATP) channel was blocked by quinine in the micromolar concentration range. The inhibition by quinine was additionally verified with the use of 86Rb+ flux experiments and submitochondrial particles. Quinine inhibited binding of the sulfonylurea derivative [3H]glibenclamide to the inner mitochondrial membrane. We conclude that quinine inhibits the cardiac mitoK(ATP) channel by acting on the mitochondrial sulfonylurea receptor.

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K + channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K ATP channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK ATP channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.

Potassium channel openers depolarize hippocampal mitochondria

We investigated the effect of the potassium channel openers diazoxide and RP66471 on mitochondrial membrane potential and mitochondrial respiration in digitonin-treated rat hippocampal homogenates. Both diazoxide and RP66471 induced a dose-dependent decrease of mitochondrial membrane potential. Concomitant with the depolarization was an increase of mitochondrial respiration. Furthermore, the mitochondrial membrane depolarization induced by diazoxide and RP66471 was significantly larger in the presence of potassium ions than in the presence of sodium ions. The diazoxide-induced (but not RP66471-induced) mitochondrial membrane depolarization was partially inhibited by blockers of the ATP-regulated potassium channel, 5-hydroxydecanoic acid or the antidiabetic sulfonylurea glibenclamide. In addition, the potassium channel openers diazoxide and RP66471 increased mitochondrial matrix volume and induced a release of cytochrome c from hippocampal mitochondria. These results indicate the presence of a mitochondrial ATP-regulated potassium channel in rat hippocampus being a target for potassium channel openers.