Mitochondrial Potassium Channels Research Articles

Single channel properties of mitochondrial large conductance potassium channel formed by BK-VEDEC splice variant

The activation of mitochondrial large conductance calcium-activated potassium (mitoBKCa) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBKCa correspond to the properties of the BKCa channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BKCa is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BKCa channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Cardioprotective Properties of Mannitol-Involvement of Mitochondrial Potassium Channels.

Cardiac preconditioning (PC) and postconditioning (PoC) are powerful measures against the consequences of myocardial ischemia and reperfusion (I/R) injury. Mannitol—a hyperosmolar solution—is clinically used for treatment of intracranial and intraocular pressure or promotion of diuresis in renal failure. Next to these clinical indications, different organ-protective properties—e.g., perioperative neuroprotection—are described. However, whether Mannitol also confers cardioprotection via a pre- and/or postconditioning stimulus, possibly reducing consequences of I/R injury, remains to be seen. Therefore, in the present study we investigated whether (1) Mannitol-induced pre- and/or postconditioning induces myocardial infarct size reduction and (2) activation of mitochondrial ATP-sensitive potassium (mKATP) channels is involved in cardioprotection by Mannitol. Experiments were performed on isolated hearts of male Wistar rats via a pressure controlled Langendorff system, randomized into 7 groups. Each heart underwent 33 min of global ischemia and 60 min of reperfusion. Control hearts (Con) received Krebs–Henseleit buffer as vehicle only. Pre- and postconditioning was achieved by administration of 11 mmol/L Mannitol for 10 min before ischemia (Man-PC) or immediately at the onset of reperfusion (Man-PoC), respectively. In further groups, the mKATP channel blocker 5HD, was applied with and without Mannitol, to determine the potential underlying cardioprotective mechanisms. Primary endpoint was infarct size, determined by triphenyltetrazolium chloride staining. Mannitol significantly reduced infarct size both as a pre- (Man-PC) and postconditioning (Man-PoC) stimulus compared to control hearts (Man-PC: 31 ± 4%; Man-PoC: 35 ± 6%, each p < 0.05 vs. Con: 57 ± 9%). The mKATP channel inhibitor completely abrogated the cardioprotective effect of Mannitol-induced pre- (5HD-PC-Man-PC: 59 ± 8%, p < 0.05 vs. Man-PC) and postconditioning (5HD-PoC-Man-PoC: 59 ± 10% vs. p < 0.05 Man-PoC). Infarct size was not influenced by 5HD itself (5HD-PC: 60 ± 14%; 5HD-PoC: 54 ± 14%, each ns vs. Con). This study demonstrates that Mannitol (1) induces myocardial pre- and postconditioning and (2) confers cardioprotection via activation of mKATP channels.

An Angiopep2-PAPTP Construct Overcomes the Blood-Brain Barrier. New Perspectives against Brain Tumors.

A developing family of chemotherapeutics—derived from 5-(4-phenoxybutoxy)psoralen (PAP-1)—target mitochondrial potassium channel mtKv1.3 to selectively induce oxidative stress and death of diseased cells. The key to their effectiveness is the presence of a positively charged triphenylphosphonium group which drives their accumulation in the organelles. These compounds have proven their preclinical worth in murine models of cancers such as melanoma and pancreatic adenocarcinoma. In in vitro experiments they also efficiently killed glioblastoma cells, but in vivo they were powerless against orthotopic glioma because they were completely unable to overcome the blood-brain barrier. In an effort to improve brain delivery we have now coupled one of these promising compounds, PAPTP, to well-known cell-penetrating and brain-targeting peptides TAT48–61 and Angiopep-2. Coupling has been obtained by linking one of the phenyl groups of the triphenylphosphonium to the first amino acid of the peptide via a reversible carbamate ester bond. Both TAT48–61 and Angiopep-2 allowed the delivery of 0.3–0.4 nmoles of construct per gram of brain tissue upon intravenous (i.v.) injection of 5 µmoles/kg bw to mice. This is the first evidence of PAPTP delivery to the brain; the chemical strategy described here opens the possibility to conjugate PAPTP to small peptides in order to fine-tune tissue distribution of this interesting compound.

Regulation of the Activity of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Through a New External Heme-Binding Site

Mitochondrial large-conductance calcium-activated potassium (mitoBKCa) channel located in the inner mitochondrial membrane is formed by the DEC splice variant of the KCNMA1 gene. The activity of mitoBKCa is regulated by many modulators, including activators such as calcium ions and inhibitors such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (–CXXCH–) located between two RCK (regulating conductance of K+) domains present in the mitochondrial matrix (internal side of the channel). Till now, there was no evidence about the existence of another heme-binding site in the mitoBKCa. However, using patch-clamp on mitoplast isolated from astrocytoma U-87MG cells and HEK293 cell line stably expressing DEC isoform of BKCa, we accidentally found that external heme also inhibits mitoBKCa suggesting the presence of yet another unknown heme-binding site. To further confirm the initial observation we used two approaches. First, we carried out patch-clamp recordings on the inner mitochondrial membrane in the outside-out configuration, and second, the pipette perfusion system was used in the inside-out configuration of the membrane patches. Both approaches allow for the application of channel modulators to the intermembrane-space (external) side of the mitoBKCa. We confirmed that hemin applied to the intermembrane space side of the channel inhibits the activity of mitoBKCa. We investigated the amino acid composition of different isoforms of BKCa and identified cysteines and histidine located at the external side of the channel, which could constitute a novel heme-binding site. We obtained HEK293 cell lines stably expressing DEC mutants of the above-mentioned cysteines and histidine. Patch-clamp experiments will be carried out to find out whether introduced mutations affect channel inhibition by external heme. This work was supported by the Polish National Science Center, grant no. 2018/31/N/NZ1/00928.

Does the Operation of Mitochondrial ATP-Dependent Potassium Channels Affect the Structural Component of Mitochondrial and Endothelial Dysfunctions in Experimental Parkinsonism?

We have previously demonstrated that the development of oxidative stress in some pathologies can be prevented by activation of the mitochondrial ATP-dependent potassium channel (mitoKATP). Here we studied the effect of modulation of mitoKATP on the development of mitochondrial and endothelial dysfunction in the medulla oblongata and myocardium of rats with experimental parkinsonism. It is known that uridine-5'-diphosphate, activator of mitoKATP, does not penetrate the plasma membrane, but it can be synthesized in cells from exogenous uridine that is delivered into cells by special transport systems. Our results suggest that mitoKATP is involved in the development of mitochondrial and endothelial dysfunction in experimental parkinsonism and prove the cardio- and neuroprotective effects of uridine.

Synthesis and pharmacological characterization of mitochondrial KATP channel openers with enhanced mitochondriotropic effects

Mitochondria play a key role for deciding fate of cells and thus are considered an attractive target for pharmacological interventions focused on containment of myocardial ischemia/reperfusion (I/R) injury. Notably, the activation of mitochondrial potassium (mitoK) channels produces a mild depolarization of mitochondrial membrane, that contributes to reduce the driving force to calcium uptake and prevents the formation of mitochondrial transition membrane pore (MPTP); these events underlie anti-ischemic cardioprotection. However, an ideal mitoK channel opener should possess the fundamental requirement to be delivered at mitochondrial level; therefore, to improve the mitochondrial delivery of a previously characterized spirocyclic benzopyrane F81, new compounds have been developed. The three original triphenylphosphonium (TPP+)-derivatives of F81 (1–3), were evaluated for their cardioprotective activity on both isolated cardiac mitochondria and cardiac H9c2 cell line. Compound 1 was further investigated in an in vivo infarct model. This work confirms that the TPP+ strategy applied to mitoKATP openers could foster mitochondrial delivery and enhance the cardioprotective effects of mitochondrial activators of potassium channels.

Участь кальційзалежних калієвих каналів великої провідності в модуляції параметрів дихання мітохондрій міокарда докозагексаєновою кислотою

Omega-3 polyunsaturated fatty acids (PUFA) provide protection against myocardial damage in ischemia-reperfusion. However, the mechanisms that provide cardioprotection are not fully understood. In this study, we investigated the effect of docosahexaenoic acid (DHA), a member of omega -3 PUFA, on mitochondrial respiration parameters and the role of mitochondrial calcium-dependent potassium channels of large conductance (ВКСа) in the implementation of these effects. Using the patch-clamp method, it was shown that functional ВКСа channels are expressed in the inner mitochondrial membrane of cardiac cells and their activity increases with the addition of DHA. We investigated the role of mitochondrial ВКСа channels in the regulation of mitochondrial respiratory processes. In experiments with isolated mitochondria from rat hearts, we showed that DHA prevented an increase in the respiratory rate of mitochondria in the V4 state and a decrease in the respiratory control elicited by addition of 10 μM Ca2+. Qualitatively the same effect was caused by NS1619, the ВКСа opener. In the presence of 10 μM Ca2+, the ВКСа channel inhibitor paxilin (1 μM) prevented the protective effect of DHA and NS1619 on the parameters of respiratory control. We conclude that mitochondrial ВКСа channels are involved in the implementation of the effects of DHA on mitochondrial respiration.

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons.

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-KATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15s reperfusion and 15s re-ischemia, and was started 30s after the 7.5min ischemic load. We recorded N-methyl-D-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca2+ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-KATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca2+ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca2+ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-KATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

Evaluating the possible role of mitochondrial ATP-sensitive potassium channels in the cardioprotective effects of morin in the isolated rat heart

AimsDuring heart ischemia, the lack of oxygen in the myocardial cells causes pH and ion disturbances and cell death through opening mitochondrial permeability transition pores (mPTP). Considering the inhibitory effects of mitochondrial ATP-dependent potassium channels (mt-KATP) on these pores and anti-ischemic effects of morin, we hypothesized that it may exert its positive effects via activating mt-KATP as well as its anti-oxidative effects. Main methodsIsolated rat hearts were perfused by Krebs-Henseleit solution enriched with the morin (0.25, 0.5 and 1 mg/L) or 5-hydroxydecanoate (5-HD, a mt-KATP blocker;100 μM) or both as needed 5 min before starting regional ischemia till the first 10 min of the reperfusion period. The reperfusion was developed with Krebs-Henseleit solution 60 or 120 min respectively for biochemical evaluations (lactate dehydrogenase and malondialdehyde level) or the assessment of myocardial infarct size. During the experiments, hemodynamic functions were recorded and cardiac arrhythmias were determined. Key findingsOur findings demonstrated that morin reduced the infarct size. Also, morin perfusion could remarkably prevent the malondialdehyde over-production during ischemia. Total ventricular ectopic beats had the same significant changes as the malondialdehyde level, in both ischemia and reperfusion phases. Morin could also relatively improve the ischemia-induced hemodynamic dysfunction. All mentioned protective effects of morin were reversed by concomitant perfusion of 5-HD. SignificanceMorin has protective effects against ischemic hearts through anti-oxidative effects. It also suggests a link between the cardioprotective effects of morin and mt-KATP. However, additional studies are required to prove this preliminary hypothesis.

Reactive Oxygen Species Mediate Nicorandil-induced Metabolic Tolerance to Spinal Cord Injury

Spinal cord injury remains a devastating complication of thoracoabdominal aortic surgery. We previously demonstrated that pretreatment with nicorandil preserved motor function in a murine spinal cord injury model through mitochondrial adenosine triphosphate-sensitive potassium channel activation. We hypothesized that the neuroprotective effect of nicorandil is mediated by downstream generation of reactive oxygen species. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Five groups were evaluated: ischemic control (n= 19); nicorandil 1.0 mg/kg (n= 17); nicorandil 1.0 mg/kg plus N acetyl L-cysteine (NAC [reactive oxygen species scavenger, n= 18)]) 150 mg/kg; NAC 150 mg/kg (n= 13); and sham (n= 10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. Mice in the sham group showed no functional deficits after surgery. Compared with ischemic control, motor function was significantly preserved in the nicorandil pretreatment group at every timepoint after ischemia. In the nicorandil plus NAC group, the motor-preserving effect of nicorandil was completely abolished (P < .001). Viable neuron quantification showed significant neuron preservation in the nicorandil group (29.± 2.6) compared with the ischemic control group (18.5 ± 2.1, P= .024) and nicorandil plus NAC group (14 ± 8.3, P= .001); no significant difference was observed between the ischemic control group and nicorandil plus NAC group (P= 0.768). Reactive oxygen species generation plays a key role in the nicorandil-induced metabolic tolerance to spinal cord injury. Manipulation of mitochondrial adenosine triphosphate-sensitive potassium channels may lead to improvement in preventing spinal cord injury after thoracoabdominal aortic interventions.

Diazoxide Protects against Myocardial Ischemia/Reperfusion Injury by Moderating ERS via Regulation of the miR-10a/IRE1 Pathway.

Nowadays, reperfusion is still the most effective treatment for ischemic heart disease. However, cardiac reperfusion therapy would lead to reperfusion injury, which may have resulted from endoplasmic reticulum stress (ERS) during reperfusion. Diazoxide (DZ) is a highly selective mitochondrial adenosine triphosphate-sensitive potassium channel opener. Its protective effect on I/R injury has been confirmed in many organs such as the heart and brain. However, the mechanism of its protective effect has not been fully elucidated. MicroRNAs (miRNAs) are widely involved in pathologies of heart disease. In this study, we found that miR-10a expression was highly upregulated in the myocardial I/R groups, and DZ treatment significantly reduced the expression of miR-10a. More importantly, we found that DZ treatment can moderate ERS via regulation of the miR-10a/IRE1 pathway in the I/R and H/R models, thereby protecting myocardial H/R injury.

Dexmedetomidine Provides Cardioprotection During Early or Late Reperfusion Mediated by Different Mitochondrial K+-Channels.

Cardioprotective interventions-such as pharmacological postconditioning-are a promising strategy to reduce deleterious consequences of ischemia and reperfusion injury (I/RI) in the heart, especially as timing and onset of myocardial infarction are unpredictable. Pharmacological postconditioning by treatment with dexmedetomidine (Dex), an α2-adrenoreceptor agonist, during reperfusion protects hearts from I/RI, independently of time point and duration of application during the reperfusion phase. The mitochondrial ATP-sensitive K (mKATP) and mitochondrial large-conductance calcium-sensitive potassium channel (mBKCa) play a pivotal role in mediating this cardioprotective effect. Therefore, we investigated whether Dex-induced cardioprotection during early or late reperfusion is mediated variously by these mitochondrial K-channels. Hearts of male Wistar rats were randomized into 8 groups and underwent a protocol of 15 minutes adaption, 33 minutes ischemia, and 60 minutes reperfusion in an in vitro Langendorff-system. A 10-minute treatment phase was started directly (first subgroup, early reperfusion) or 30 minutes (second subgroup, late reperfusion) after the onset of reperfusion. Control (Con) hearts received vehicle only. In the first subgroup, hearts were treated with 3 nM Dex, 100 µM mKATP-channel blocker 5-hydroxydecanoate (5HD) or 1 µM mBKCa-channel blocker Paxilline (Pax) alone or with respective combinations (5HD + Dex, Pax + Dex). Hearts of the second subgroup received Dex alone (Dex30') or in combination with the respective blockers (5HD + Dex30', Pax + Dex30'). Infarct size was determined with triphenyltetrazoliumchloride staining. Hemodynamic variables were recorded during the whole experiment. During early reperfusion (first subgroup), the infarct size reducing effect of Dex (Con: 57% ± 9%, Dex: 31% ± 7%; P< .0001 versus Con) was completely abolished by 5HD and Pax (52% ± 6%; Pax + Dex: 53% ± 4%; each P< .0001 versus Dex), while both blockers alone had no effect on infarct size (5HD: 54% ± 8%, Pax: 53% ± 11%). During late reperfusion (second subgroup) the protective effect of Dex (Dex30': 33% ± 10%, P< .0001 versus Con) was fully abrogated by Pax (Pax + Dex30': 58% ± 7%, P < .0001 versus Dex30'), whereas 5HD did not block cardioprotection (5HD + Dex30': 36% ± 7%). Between groups and within each group throughout reperfusion no significant differences in hemodynamic variables were detected. Cardioprotection by treatment with Dex during early reperfusion seems to be mediated by both mitochondrial K-channels, whereas during late reperfusion only mBKCa-channels are involved.

Mitochondrial Potassium Channels as Druggable Targets.

Mitochondrial potassium channels have been described as important factors in cell pro-life and death phenomena. The activation of mitochondrial potassium channels, such as ATP-regulated or calcium-activated large conductance potassium channels, may have cytoprotective effects in cardiac or neuronal tissue. It has also been shown that inhibition of the mitochondrial Kv1.3 channel may lead to cancer cell death. Hence, in this paper, we examine the concept of the druggability of mitochondrial potassium channels. To what extent are mitochondrial potassium channels an important, novel, and promising drug target in various organs and tissues? The druggability of mitochondrial potassium channels will be discussed within the context of channel molecular identity, the specificity of potassium channel openers and inhibitors, and the unique regulatory properties of mitochondrial potassium channels. Future prospects of the druggability concept of mitochondrial potassium channels will be evaluated in this paper.

The mitochondrial K-ATP channel opener diazoxide upregulates STIM1 and Orai1 via ROS and the MAPK pathway in adult rat cardiomyocytes

BackgroundOpeners of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia–reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1.ResultsQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFκB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression.ConclusionsOur results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.

Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage.

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at −40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor α (TNF-α) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

Signaling pathways targeting mitochondrial potassium channels.

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca2+-activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels.

A new brain mitochondrial sodium-sensitive potassium channel: effect of sodium ions on respiratory chain activity.

We have determined the electropharmacological properties of a new potassium channel from brain mitochondrial membrane using a planar lipid bilayer method. Our results show the presence of a channel with a conductance of 150 pS at potentials between 0 and -60 mV in 200 mM cis/50 mM trans KCl solutions. The channel was voltage independent, with an open probability value of approximately 0.6 at different voltages. ATP did not affect current amplitude or open probability at positive and negative voltages. Notably, adding iberiotoxin, charybdotoxin, lidocaine or margatoxin had no effect on the channel behavior. Similarly, no changes were observed by decreasing the cis pH to 6. Interestingly, the channel was inhibited by adding sodium in a dose-dependent manner. Our results also indicated a significant increase in mitochondrial complex IV activity and membrane potential and a decrease in complex I activity and mitochondrial ROS production in the presence of sodium ions. We propose that inhibition of mitochondrial potassium transport by sodium ions on potassium channel opening could be important for cell protection and ATP synthesis.

The Melatonin Receptor Agonist Ramelteon Induces Cardioprotection that Requires MT2 Receptor Activation and Release of Reactive Oxygen Species

PurposeThe melatonin receptor (MT) agonist ramelteon has a higher affinity to MT1 than for MT2 receptors and induces cardioprotection by involvement of mitochondrial potassium channels. Activation of mitochondrial potassium channels leads to release of free radicals. We investigated whether (1) ramelteon-induced cardioprotection is MT2 receptor specific and (2) if free radicals are involved in ramelteon-induced cardioprotection.MethodsHearts of male Wistar rats were randomized, placed on a Langendorff system, and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia hearts were perfused with ramelteon (Ram) with or without the MT2 receptor inhibitor 4-phenyl-2-propionamidotetralin (4P-PDOT+Ram, 4P-PDOT). In subsequent experiments, ramelteon was administered together with the radical oxygen species (ROS) scavenger N-2-mercaptopropionylglycine (MPG+Ram). To determine whether the blockade of ramelteon-induced cardioprotection can be restored, we combined ramelteon and MPG with mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A (CsA) at different time points. Infarct size was determined by triphenyltetrazolium chloride (TTC) staining.ResultsRamelteon-induced infarct size reduction was completely blocked by 4P-PDOT and MPG. Ramelteon and MPG combined with CsA before ischemia were not cardioprotective but CsA at the onset of reperfusion could restore infarct size reduction.ConclusionsThis study shows for the first time that despite the higher affinity to MT1 receptors, (1) ramelteon-induced cardioprotection involves MT2 receptors, (2) cardioprotection requires ROS release, and (3) inhibition of the mPTP can restore infarct size reduction.

Beneficial Effect of Citrus Flavonoid - Naringenin on Endothelial Cells by Activation of Mitochondrial Potassium Channels

Beneficial Effect of Citrus Flavonoid - Naringenin on Endothelial Cells by Activation of Mitochondrial Potassium Channels

Development of a new simple mitochondria-targeted fluorescent K+ sensor and the application in high-throughput monitoring K+ fluxes

A new simple mitochondria-targeted fluorescent potassium ion (K+) sensor (NK1) was synthesized and investigated. The synthetic route of this sensor was much more concise than the previously reported mitochondrial-targeting KS6, enabling the potential broad applications of this sensor NK1. NK1 displays good responses to K+ with broad detection range from 10 to 500 mM, short response time (0.2 s), high sensitivity with 160 folds’ fluorescence enhancement at 1000 mM K+, a large dissociation constant (Kd about 200 mM), and minimum responses to physiological pH (5.0–9.0) as well as other biologically relevant metal ions. K+ fluxes (efflux and influx) in the mitochondria of living cells were observed upon manipulation with ionophores (nigericin or ionomycin) and various concentrations of KCl both under confocal microscope and especially microplate reader for first time high-throughput in-situ monitoring of mitochondrial K+ fluxes. The development of easily synthesized NK1 with excellent performance will be greatly beneficial for ion channel relative drug screening and the investigation of mitochondrial potassium channels as well as mitochondrial metabolism.