Mitochondrial Potassium Channels Research Articles

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

Flavonoids as new regulators of mitochondrial potassium channels: contribution to cardioprotection.

Acute myocardial ischemia is one of the major causes of illness in western society. Reduced coronary blood supply leads to cell death and loss of cardiomyocyte population, resulting in serious and often irreversible consequences on myocardial function. Mitochondrial potassium (mitoK) channels have been identified as fine regulators of mitochondrial function and, consequently, in the metabolism of the whole cell, and in the mechanisms underlying the cardioprotection. Interestingly, mitoK channels represent a novel putative target for treating cardiovascular diseases, particularly myocardial infarction, and their modulators represent an interesting tool for pharmacological intervention. In this review, we took up the challenge of selecting flavonoids that show cardioprotective properties through the activation of mitoK channels. A brief overview of the main information on mitoK channels and their participation in the induction of cytoprotective processes was provided. Then, naringenin, quercetin, morin, theaflavin, baicalein, epigallocatechin gallate, genistein, puerarin, luteolin and proanthocyanidins demonstrated to be effective modulators of mitoK channels activity, mediating many beneficial effects. The pathophysiological role of mitoK channels has been investigated as well as the impact of flavonoids on this target with particular attention to their potential role in the prevention of cardiovascular disorders.

External Hemin as an Inhibitor of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Activity.

The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is located in the inner mitochondrial membrane and seems to play a crucial role in cytoprotection. The mitoBKCa channel 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 domains present in the mitochondrial matrix. In the present study, we used the patch-clamp technique in the outside-out configuration to record the activity of mitoBKCa channels. This allowed for the application of channel modulators to the intermembrane-space side of the mitoBKCa. We found that hemin applied in this configuration inhibits the activity of mitoBKCa. In addition, we proved that the observed hemin effect is specific and it is not due to its interaction with the inner mitochondrial membrane. Our data suggest the existence of a new potential heme/hemin binding site in the structure of the mitoBKCa channel located on the mitochondrial intermembrane space side, which could constitute a new way for the regulation of mitoBKCa channel activity.

BKCa Activator NS1619 Improves the Structure and Function of Skeletal Muscle Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary disease caused by the absence of the dystrophin protein. This is secondarily accompanied by a dysregulation of ion homeostasis, in which mitochondria play an important role. In the present work, we show that mitochondrial dysfunction in the skeletal muscles of dystrophin-deficient mdx mice is accompanied by a reduction in K+ transport and a decrease in its content in the matrix. This is associated with a decrease in the expression of the mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) in the muscles of mdx mice, which play an important role in cytoprotection. We observed that the BKCa activator NS1619 caused a normalization of mitoBKCa expression and potassium homeostasis in the muscle mitochondria of these animals, which was accompanied by an increase in the calcium retention capacity, mitigation of oxidative stress, and improvement in mitochondrial ultrastructure. This effect of NS1619 contributed to the reduction of degeneration/regeneration cycles and fibrosis in the skeletal muscles of mdx mice as well as a normalization of sarcomere size, but had no effect on the leakage of muscle enzymes and muscle strength loss. In the case of wild-type mice, we noted the negative effect of NS1619 manifested in the inhibition of the functional activity of mitochondria and disruption of their structure, which, however, did not significantly affect the state of the skeletal muscles of the animals. This article discusses the role of mitoBKCa in the development of DMD and the prospects of the approach associated with the correction of its function in treatments of this secondary channelopathy.

Luteolin-Induced Activation of Mitochondrial BKCa Channels: Undisclosed Mechanism of Cytoprotection.

Luteolin (LUT) is a well-known flavonoid that exhibits a number of beneficial properties. Among these, it shows cardioprotective effects, as confirmed by numerous studies. However, its effect on mitochondrial potassium channels, the activation of which is related to cytoprotection, as well as on heart ischemia/reperfusion (I/R) damage prevention, has not yet been investigated. The large conductance calcium-regulated potassium channel (mitoBKCa) has been identified in both the mitochondria of the vascular endothelial cells, which plays a significant role in the functioning of the cardiovascular system under oxidative stress-related conditions, and in the mitochondria of cardiomyocytes, where it is deeply involved in cardiac protection against I/R injury. Therefore, the aim of this study was to explore the role of the mitoBKCa channel in luteolin-induced cytoprotection. A number of in vitro, in vivo, ex vivo and in silico studies have confirmed that luteolin activates this channel in the mitochondria of cardiomyocytes and endothelial cells, which in turn leads to the protection of the endothelium and a significant reduction in the extent of damage resulting from myocardial infarction, where this effect was partially abolished by the mitoBKCa channel blocker paxilline. In conclusion, these results suggest that luteolin has cardioprotective effects, at least in part, through the activation of the mitoBKCa channel, shedding light on a new putative mechanism of action.

Cardioprotective Effects of Grapefruit IntegroPectin Extracted via Hydrodynamic Cavitation from By-Products of Citrus Fruits Industry: Role of Mitochondrial Potassium Channels

Citrus flavonoids are well-known for their beneficial effects at the cardiovascular and cardio-metabolic level, but often the encouraging in vitro results are not confirmed by in vivo approaches; in addition, the clinical trials are also inconsistent. Their limited bioavailability can be, at least in part, the reason for these discrepancies. Therefore, many efforts have been made towards the improvement of their bioavailability. Hydrodynamic cavitation methods were successfully applied to the extraction of byproducts of the Citrus fruits industry, showing high process yields and affording stable phytocomplexes, known as IntegroPectin, endowed with great amounts of bioactive compounds and high water solubility. The cardioprotective effects of grapefruit IntegroPectin were evaluated by an ex vivo ischemia/reperfusion protocol. Further pharmacological characterization was carried out to assess the involvement of mitochondrial potassium channels. Grapefruit IntegroPectin, where naringin represented 98% of the flavonoids, showed anti-ischemic cardioprotective activity, which was better than pure naringenin (the bioactive aglycone of naringin). On cardiac-isolated mitochondria, this extract confirmed that naringenin/naringin were involved in the activation of mitochondrial potassium channels. The hydrodynamic cavitation-based extraction confirmed a valuable opportunity for the exploitation of Citrus fruits waste, with the end product presenting high levels of Citrus flavonoids and improved bioaccessibility that enhances its nutraceutical and economic value.

Role of miR-133/Dio3 Axis in the T3-Dependent Modulation of Cardiac mitoK-ATP Expression.

The opening of the ATP-sensitive mitochondrial potassium channel (mitok-ATP) is a common goal of cardioprotective strategies in the setting of acute and chronic myocardial disease. The biologically active thyroid hormone (TH), 3-5-3-triiodothyronine (T3), has been indicated as a potential activator of mitoK-ATP but the underlying mechanisms are still elusive. Here we describe a novel role of T3 in the transcriptional regulation of mitoK and mitoSur, the recently identified molecular constituents of the channel. To mimic human ischemic heart damage, we used a rat model of a low T3 state as the outcome of a myocardial ischemia/reperfusion event, and neonatal rat cardiomyocytes (NRCM) challenged with hypoxia or H2O2. Either in the in vivo or in vitro models, T3 administration to recover the physiological concentrations was able to restore the expression level of both the channel subunits, which were found to be downregulated under the stress conditions. Furthermore, the T3-mediated transcriptional activation of mitoK-ATP in the myocardium and NRCM was associated with the repression of the TH-inactivating enzyme, deiodinase 3 (Dio3), and an up-regulation of the T3-responsive miR-133a-3p. Mechanistically, the loss and gain of function experiments and reporter gene assays performed in NRCM, have revealed a new regulatory axis whereby the silencing of Dio3 under the control of miR-133a-3p drives the T3-dependent modulation of cardiac mitoK and mitoSur transcription.

Current Challenges of Mitochondrial Potassium Channel Research.

In this paper, the current challenges of mitochondrial potassium channels research were critically reviewed. Even though recent progress in understanding K+ traffic in mitochondria has been substantial, some basic issues of this process remain unresolved. Here, we focused on the critical discussion of the molecular identity of various mitochondrial potassium channels. This point helps to clarify why there are different potassium channels in specific mitochondria. We also described interactions of mitochondrial potassium channel subunits with other mitochondrial proteins. Posttranslational modifications of mitochondrial potassium channels and their import are essential but unexplored research areas. Additionally, problems with the pharmacological targeting of mitochondrial potassium channel were illustrated. Finally, the limitation of the techniques used to measure mitochondrial potassium channels was explained. We believe that recognizing these problems may be interesting for readers but will also help to progress the field of mitochondrial potassium channels.

Inhibition of a Mitochondrial Potassium Channel in Combination with Gemcitabine and Abraxane Drastically Reduces Pancreatic Ductal Adenocarcinoma in an Immunocompetent Orthotopic Murine Model.

Simple SummaryTreatment of pancreas ductal adenocarcinoma (PDAC) remains challenging due to the late stage of presentation, limited efficacy of cytotoxic chemotherapies, and aggressive tumor biology. Novel therapeutic targets are desperately needed. The voltage-gated potassium channel, Kv1.3, is one such unique target. It has been extensively studied in many cancers but less is known in pancreas cancer. In this study, we evaluated the tissue expression of Kv1.3 in resected PDAC and tumor inhibition using novel Kv1.3 inhibitors developed by our group (PCARBTP and PAPTP) with cytotoxic chemotherapies. We found that Kv1.3 is expressed in early stage, non-metastatic, resectable pancreas cancer specimens. Treatment with novel mitochondrial Kv1.3 inhibitors resulted in 95% reduced tumor growth when combined with cytotoxic chemotherapies. This near complete eradication of tumors using this treatment strategy shows that Kv1.3 represents an innovative therapeutic target for pancreas cancer therapy.Pancreas ductal adenocarcinoma (PDAC) is one the most aggressive cancers and associated with very high mortality, requiring the development of novel treatments. The mitochondrial voltage-gated potassium channel, Kv1.3 is emerging as an attractive oncologic target but its function in PDAC is unknown. Here, we evaluated the tissue expression of Kv1.3 in resected PDAC from 55 patients using immunohistochemistry (IHC) and show that all tumors expressed Kv1.3 with 60% of tumor specimens having high Kv1.3 expression. In Pan02 cells, the recently developed mitochondria-targeted Kv1.3 inhibitors PCARBTP and PAPTP strongly reduced cell survival in vitro. In an orthotopic pancreas tumor model (Pan02 cells injected into C57BL/6 mice) in immune-competent mice, injection of PAPTP or PCARBTP resulted in tumor reductions of 87% and 70%, respectively. When combined with clinically used Gemcitabine plus Abraxane (albumin-bound paclitaxel), reduction reached 95% and 80% without resultant organ toxicity. Drug-mediated tumor cell death occurred through the p38-MAPK pathway, loss of mitochondrial membrane potential, and oxidative stress. Resistant Pan02 clones to PCARBTP escaped cell death through upregulation of the antioxidant system. In contrast, tumor cells did not develop resistance to PAPTP. Our data show that Kv1.3 is highly expressed in resected human PDAC and the use of novel mitochondrial Kv1.3 inhibitors combined with cytotoxic chemotherapies might be a novel, effective treatment for PDAC.

Flavonoid quercetin abolish paxilline inhibition of the mitochondrial BKCa channel

Large-conductance calcium-regulated potassium channel (BKCa) is known to play an important role in physiological and pathological processes. Despite the BKCa channel being encoded by one gene, this channel has been found to be located not only in the cell membrane but also in the membranes of intracellular compartments, such as in the inner mitochondrial membrane. With some differences, the mitochondrial BKCa (mitoBKCa) channel has been shown to be activated or inhibited by both synthetic and natural compounds. One of them, paxilline, has been considered to be a canonical blocker of this channel.In the previous study, we showed that the natural origin substance quercetin activates the mitoBKCa channel at ten times lower the concentration compared to channel present in the plasma membrane. Here, using the patch-clamp technique, we report that after inhibition of mitoBKCa channels by paxilline, quercetin activates these channels, indicating a paxilline and quercetin binding competition in the regulation of the mitoBKCa channel. To support our hypothesis, we used an analog of quercetin – isorhamnetin, a substance with one substituent changed. Isorhamnetin has no effect on the mitoBKCa channel activity, and after its application, paxilline fully inhibits the channel. Additionally, the molecular modeling studies were used. The results of docking quercetin and paxilline to the BKCa channel suggest that paxilline cannot bind after activation of the channel with quercetin. It seems that the likely mechanism of this phenomenon is the formation of spatial hindrance by quercetin. The results obtained shed a completely new, groundbreaking in the paxilline context, light on the current knowledge about mitochondrial potassium channel regulation.

Targeting Mitochondrial Large-Conductance Calcium-Activated Potassium Channel by Hydrogen Sulfide via Heme-Binding Site.

Reperfusion together with the preceding ischemic period results in serious damage to brain and heart tissues. Activation of potassium channels from the inner mitochondrial membrane leads to cytoprotection during such events. The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is one of these cytoprotective channels. It was previously shown that BKCa channels are blocked by hemin, which is present in excess during hemorrhage. In the experiments described in this work, we checked whether NaHS, known as a donor of gasotransmitter hydrogen sulfide (H2S), which can play an important role in cytoprotection, interacts with mitoBKCa channels. Indeed, using the biotin-switch method, it was found that mitoBKCa channels undergo S-sulfhydration in the presence of NaHS. Although patch-clamp experiments showed that NaHS has negligible effects on the activity of mitoBKCa channels, NaHS has been shown to almost fully activate hemin-inhibited mitoBKCa channels. The effects of NaHS were mimicked by imidazole, suggesting a common mechanism of activation of mitoBKCa channels inhibited by heme/hemin by molecules able to coordinate the iron ion of porphyrin. A set of absorption spectroscopy experiments with the 23 amino acid model peptides containing the heme-binding motif CXXCH suggested previously unrecognized roles of cysteines in heme binding. SIGNIFICANCE STATEMENT: The activity of mitochondrial channels including mitoBKCa seems to play a significant role in cytoprotection during ischemia/reperfusion. Hemin, which is present in excess during hemorrhage, can potentially bind to and inhibit mitoBKCa activity. We found that hydrogen sulfide does not affect mitoBKCa activity unless it is blocked by hemin. In this case, hydrogen sulfide activates hemin-inhibited mitoBKCa by binding to hemin iron. The hydrogen sulfide effect could be mimicked in patch-clamp experiments by imidazole probably acting by a similar mechanism.

Characterization of the pathophysiological roles of a calcium-dependent potassium channel in mitochondria

The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is functional in the inner mitochondrial membrane (IMM) of various cancer cell lines, as demonstrated by patch-clamp and Western Blot. Decreased expression of KCa3.1 was found to affect oxidative phosphorylation and ATP production (Kovalenko et al., 2016) suggesting a role in fine-tuning mitochondrial energy metabolism, but its precise role in the IMM has not been elucidated so far. Here we report the synthesis and characterization of a mitochondria-targeted derivative of a high-affinity KCa3.1 antagonist.

The 40-Hz White Light-Emitting Diode (LED) Improves the Structure-Function of the Brain Mitochondrial KATP Channel and Respiratory Chain Activities in Amyloid Beta Toxicity.

It has been described that using noninvasive exposure to 40-Hz white light LED reduces amyloid-beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). However, the mechanisms remain to be identified. Since AD impairs mitochondrial potassium channels and respiratory chain activity, the objectives of the current study were to determine the effect of 40-Hz white light LED on structure-function of mitoKATP channel and brain mitochondrial respiratory chain activity, production of reactive oxygen species (ROS), and ΔΨm in AD. Single mitoKATP channel was considered using a channel incorporated into the bilayer lipid membrane and expression of mitoKATP-Kir6.1 subunit as a pore-forming subunit of the channel was determined using a western blot analysis in Aβ1-42 toxicity and light-treated rats. Our results indicated a severe decrease in mito-KATP channel permeation and Kir6.1 subunit expression coming from the Aβ1-42-induced neurotoxicity. Furthermore, we found that Aβ1-42-induced neurotoxicity decreased activities of complexes I and IV and increased ROS production and ΔΨm. Surprisingly, light therapy increased channel permeation and mitoKATP-Kir6.1 subunit expression. Noninvasive 40-Hz white light LED treatment also increased activities of complexes I and IV and decreased ROS production and ΔΨm up to ~ 70%. Here, we report that brain mito-KATP channel and respiratory chain are, at least in part, novel targets of 40-Hz white light LED therapy in AD.

Methods of Measuring Mitochondrial Potassium Channels: A Critical Assessment.

In this paper, the techniques used to study the function of mitochondrial potassium channels are critically reviewed. The majority of these techniques have been known for many years as a result of research on plasma membrane ion channels. Hence, in this review, we focus on the critical evaluation of techniques used in the studies of mitochondrial potassium channels, describing their advantages and limitations. Functional analysis of mitochondrial potassium channels in comparison to that of plasmalemmal channels presents additional experimental challenges. The reliability of functional studies of mitochondrial potassium channels is often affected by the need to isolate mitochondria and by functional properties of mitochondria such as respiration, metabolic activity, swelling capacity, or high electrical potential. Three types of techniques are critically evaluated: electrophysiological techniques, potassium flux measurements, and biochemical techniques related to potassium flux measurements. Finally, new possible approaches to the study of the function of mitochondrial potassium channels are presented. We hope that this review will assist researchers in selecting reliable methods for studying, e.g., the effects of drugs on mitochondrial potassium channel function. Additionally, this review should aid in the critical evaluation of the results reported in various articles on mitochondrial potassium channels.

Alternative Targets for Modulators of Mitochondrial Potassium Channels.

Mitochondrial potassium channels control potassium influx into the mitochondrial matrix and thus regulate mitochondrial membrane potential, volume, respiration, and synthesis of reactive oxygen species (ROS). It has been found that pharmacological activation of mitochondrial potassium channels during ischemia/reperfusion (I/R) injury activates cytoprotective mechanisms resulting in increased cell survival. In cancer cells, the inhibition of these channels leads to increased cell death. Therefore, mitochondrial potassium channels are intriguing targets for the development of new pharmacological strategies. In most cases, however, the substances that modulate the mitochondrial potassium channels have a few alternative targets in the cell. This may result in unexpected or unwanted effects induced by these compounds. In our review, we briefly present the various classes of mitochondrial potassium (mitoK) channels and describe the chemical compounds that modulate their activity. We also describe examples of the multidirectional activity of the activators and inhibitors of mitochondrial potassium channels.

Mediation of the Cardioprotective Effects of Mannitol Discovered, with Refutation of Common Protein Kinases.

The osmodiuretic agent Mannitol exerts cardioprotection against ischemia and reperfusion (I/R) injury when applied as a pre- and/or postconditioning stimulus. Previously, we demonstrated that these properties are mediated via the activation of mitochondrial ATP-sensitive potassium (mKATP) channels. However, considering Mannitol remains in the extracellular compartment, the question arises as to which receptor and intracellular signaling cascades are involved in myocardial protection by the osmodiuretic substance. Protein kinase B (Akt) and G (PKG), as part of the reperfusion injury salvage kinase (RISK) and/or endothelial nitric oxide (eNOS)/PKG pathway, are two well-investigated intracellular targets conferring myocardial protection upstream of mitochondrial potassium channels. Adenosine receptor subtypes have been shown to trigger different cardioprotective pathways, for example, the reperfusion injury. Further, Mannitol induces an increased activation of the adenosine 1 receptor (A1R) in renal cells conferring its nephroprotective properties. Therefore, we investigated whether (1) Akt and PKG are possible signaling targets involved in Mannitol-induced conditioning upstream of the mKATP channel and/or whether (2) cardioprotection by Mannitol is mediated via activation of the A1R. All experiments were performed on male Wistar rats in vitro employing the Langendorff isolated heart perfusion technique with infarct size determination as the primary endpoint. To unravel possible protein kinase activation, Mannitol was applied in combination with the Akt (MK2206) or PKG (KT5823) inhibitor. In further groups, an A1R blocker (DPCPX) was given with or without Mannitol. Preconditioning with Mannitol (Man) significantly reduced the infarct size compared to the control group. Co-administration of the A1R blocker DPXPC fully abolished myocardial protection of Mannitol. Interestingly and in contrast to the initial hypothesis, neither administration of the Akt nor the PKG blocker had any impact on the cardioprotective properties of Mannitol-induced preconditioning. These results are quite unexpected and show that the protein kinases Akt and PKG—as possible targets of known protective signaling cascades—are not involved in Mannitol-induced preconditioning. However, the cardioprotective effects of Mannitol are mediated via the A1R.

Uridine treatment prevents myocardial injury in rat models of acute ischemia and ischemia/reperfusion by activating the mitochondrial ATP-dependent potassium channel

The effect of uridine on the myocardial ischemic and reperfusion injury was investigated. A possible mechanism of its cardioprotective action was established. Two rat models were used: (1) acute myocardial ischemia induced by occlusion of the left coronary artery for 60 min; and (2) myocardial ischemia/reperfusion with 30-min ischemia and 120-min reperfusion. In both models, treatment with uridine (30 mg/kg) prevented a decrease in cell energy supply and in the activity of the antioxidant system, as well as an increase in the level of lipid hydroperoxides and diene conjugates. This led to a reduction of the necrosis zone in the myocardium and disturbances in the heart rhythm. The blocker of the mitochondrial ATP-dependent potassium (mitoKATP) channel 5-hydroxydecanoate limited the positive effects of uridine. The data indicate that the cardioprotective action of uridine may be related to the activation of the mitoKATP channel. Intravenously injected uridine was more rapidly eliminated from the blood in hypoxia than in normoxia, and the level of the mitoKATP channel activator UDP in the myocardium after uridine administration increased. The results suggest that the use of uridine can be a potentially effective approach to the management of cardiovascular diseases.

Cytoprotective effects of the flavonoid quercetin by activating mitochondrial BKCa channels in endothelial cells.

Mitochondrial potassium channels have been implicated in cytoprotective mechanisms. Activation of the mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channel is important for protecting brain tissue against stroke damage as well as heart tissue against ischemia damage. In this paper, we examine the effect of the natural flavonoid quercetin as an activator of the mitoBKCa channel. Quercetin has a beneficial effect on many processes in the human body and interacts with many receptors and signaling pathways. We found that quercetin acts on mitochondria as a mitoBKCa channel opener. The activation observed with the patch-clamp technique was potent and increased the channel open probability from approximately 0.35 to 0.95 at + 40 mV in the micromolar concentration range. Moreover, quercetin at a concentration of 10 µM protected cells by reducing damage from treatment factors (tumor necrosis factor α and cycloheximide) by 40%, enhancing cellular migration and depolarizing the mitochondrial membrane. Moreover, the presence of quercetin increased the gene expression and protein level of the mitoBKCa β3 regulatory subunit. The observed cytoprotective effects suggested the involvement of BKCa channel activation. Additionally, the newly discovered mitoBKCa activator quercetin elucidates a new mitochondrial pathway that is beneficial for vascular endothelial cells.

Multidimensional Regulation of Cardiac Mitochondrial Potassium Channels.

Mitochondria play a fundamental role in the energetics of cardiac cells. Moreover, mitochondria are involved in cardiac ischemia/reperfusion injury by opening the mitochondrial permeability transition pore which is the major cause of cell death. The preservation of mitochondrial function is an essential component of the cardioprotective mechanism. The involvement of mitochondrial K+ transport in this complex phenomenon seems to be well established. Several mitochondrial K+ channels in the inner mitochondrial membrane, such as ATP-sensitive, voltage-regulated, calcium-activated and Na+-activated channels, have been discovered. This obliges us to ask the following question: why is the simple potassium ion influx process carried out by several different mitochondrial potassium channels? In this review, we summarize the current knowledge of both the properties of mitochondrial potassium channels in cardiac mitochondria and the current understanding of their multidimensional functional role. We also critically summarize the pharmacological modulation of these proteins within the context of cardiac ischemia/reperfusion injury and cardioprotection.

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca2+-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BKCa channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBKCa channel in a bronchial epithelial cell line were described.