Loss of the large conductance calcium-activated potassium channel causes an increase in mitochondrial reactive oxygen species in glioblastoma cells.

Mitochondrial potassium (mitoK) channels play an important role in cellular physiology. These channels are expressed in healthy tissues and cancer cells. Activation of mitoK channels can protect neurons and cardiac tissue against injury induced by ischemia-reperfusion. In cancer cells, inhibition of mitoK channels leads to an increase in mitochondrial reactive oxygen species, which leads to cell death. In glioma cell activity of the mitochondrial, large conductance calcium-activated potassium (mitoBKCa) channel is regulated by the mitochondrial respiratory chain. In our project, we used CRISPR/Cas9 technology in human glioblastoma U-87 MG cells to generate knockout cell lines lacking the α-subunit of the BKCa channel encoded by the KCNMA1 gene, which also encodes cardiac mitoBKCa. Mitochondrial patch-clamp experiments showed the absence of an active mitoBKCa channel in knockout cells. Additionally, the absence of this channel resulted in increased levels of mitochondrial reactive oxygen species. However, analysis of the mitochondrial respiration rate did not show significant changes in oxygen consumption in the cell lines lacking BKCa channels compared to the wild-type U-87 MG cell line. These observations were reflected in the expression levels of selected mitochondrial genes, organization of the respiratory chain, and mitochondrial morphology, which did not show significant differences between the analyzed cell lines. In conclusion, we show that in U-87 MG cells, the pore-forming subunit of the mitoBKCa channel is encoded by the KCNMA1 gene. Additionally, the presence of this channel is important for the regulation of reactive oxygen species levels in mitochondria.

To what extent naringenin binding and membrane depolarization shape mitoBK channel gating-A machine learning approach.

The large conductance voltage- and Ca2+-activated K+ channels from the inner mitochondrial membrane (mitoBK) are modulated by a number of factors. Among them flavanones, including naringenin (Nar), arise as a promising group of mitoBK channel regulators from a pharmacological point of view. It is well known that in the presence of Nar the open state probability (pop) of mitoBK channels significantly increases. Nevertheless, the molecular mechanism of the mitoBK-Nar interactions remains still unrevealed. It is also not known whether the effects of naringenin administration on conformational dynamics can resemble those which are exerted by the other channel-activating stimuli. In aim to answer this question, we examine whether the dwell-time series of mitoBK channels which were obtained at different voltages and Nar concentrations (yet allowing to reach comparable pops) are discernible by means of artificial intelligence methods, including k-NN and shapelet learning. The obtained results suggest that the structural complexity of the gating dynamics is shaped both by the interaction of channel gate with the voltage sensor (VSD) and the Nar-binding site. For a majority of data one can observe stimulus-specific patterns of channel gating. Shapelet algorithm allows to obtain better prediction accuracy in most cases. Probably, because it takes into account the complexity of local features of a given signal. About 30% of the analyzed time series do not sufficiently differ to unambiguously distinguish them from each other, which can be interpreted in terms of the existence of the common features of mitoBK channel gating regardless of the type of activating stimulus. There exist long-range mutual interactions between VSD and the Nar-coordination site that are responsible for higher levels of Nar-activation (Δpop) at deeply depolarized membranes. These intra-sensor interactions are anticipated to have an allosteric nature.

The cross-correlation-based analysis to digest the conformational dynamics of the mitoBK channels in terms of their modulation by flavonoids.

The activity of mitochondrial large-conductance voltage- and [Formula: see text]-activated [Formula: see text] channels (mitoBK) is regulated by a number of biochemical factors, including flavonoids. In particular, naringenin (Nar) and quercetin (Que) reached reasonable scientific attention due to their well-pronounced channel-activating effects. The open-reinforcing outcomes of Nar and Que on the mitoBK channel gating have been already reported. Nevertheless, the molecular picture of the corresponding channel-ligand interactions remains still to be revealed. In this work, we investigate the effects of the Nar and Que on the conformational dynamics of the mitoBK channel. In this aim, the cross-correlation-based analysis of the single-channel signals recorded by the patch-clamp method is performed. The obtained results in the form of phase space diagrams enable us to visually monitor the effects exerted by the considered flavonoids at the level of temporal characteristics of repetitive sequences of channel conformations. It turns out that the mitoBK channel activation by naringenin and quercetin does not lead to the change in the number of clusters within the phase space diagrams, which can be related to the constant number of available channel macroconformations regardless of the flavonoid administration. The localization and occupancy of the clusters of cross-correlated sequences suggest that mitoBK channel stimulation by flavonoids affects the relative stability of channel conformations and the kinetics of switching between them. For most clusters, greater net effects are observed in terms of quercetin administration in comparison with naringenin. It indicates stronger channel interaction with Que than Nar.

Mitochondrial potassium channels: A novel calcitriol target.

BACKGROUND: Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. METHODS: The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. RESULTS: For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. CONCLUSIONS: Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

Effect of Quercetin on mitoBKCa Channel and Mitochondrial Function in Human Bronchial Epithelial Cells Exposed to Particulate Matter.

Particulate matter (PM) exposure increases reactive oxygen species (ROS) levels. It can lead to inflammatory responses and damage of the mitochondria thus inducing cell death. Recently, it has been shown that potassium channels (mitoK) located in the inner mitochondrial membrane are involved in cytoprotection, and one of the mechanisms involves ROS. To verify the cytoprotective role of mitoBKCa, we performed a series of experiments using a patch-clamp, transepithelial electrical resistance assessment (TEER), mitochondrial respiration measurements, fluorescence methods for the ROS level and mitochondrial membrane potential assessment, and cell viability measurements. In the human bronchial epithelial cell model (16HBE14σ), PM < 4 µm in diameter (SRM-PM4.0) was used. We observed that PM decreased TEER of HBE cell monolayers. The effect was partially abolished by quercetin, a mitoBKCa opener. Consequently, quercetin decreased the mitochondrial membrane potential and increased mitochondrial respiration. The reduction of PM-induced ROS level occurs both on cellular and mitochondrial level. Additionally, quercetin restores HBE cell viability after PM administration. The incubation of cells with PM substantially reduced the mitochondrial function. Isorhamnetin had no effect on TEER, the mitoBKCa activity, respiratory rate, or mitochondrial membrane potential. Obtained results indicate that PM has an adverse effect on HBE cells at the cellular and mitochondrial level. Quercetin is able to limit the deleterious effect of PM on barrier function of airway epithelial cells. We show that the effect in HBE cells involves mitoBKCa channel-activation. However, quercetin's mechanism of action is not exclusively determined by modulation of the channel activity.

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.

Regulation of Lipid Bilayer Ion Permeability by Antibacterial Polymethyloxazoline-Polyethyleneimine Copolymers.

Amphiphilic antimicrobial polymers display activity against the outer bacterial cell membrane, triggering various physiological effects. We investigated the regulation of ion transport across the lipid bilayer to understand differences in biological activity for a series of amphiphilic polymethyloxazoline - polyethyleneimine copolymers. The results confirmed that the tested structures were able to increase the permeability of the lipid bilayer (LB) membrane or its rupture. Black lipid membrane (BLM) experiments show that the triggered conductance profile and its character is strongly correlated with the polymer structure and zeta potential. The polymer exhibiting the highest antimicrobial activity promotes ion transport by using a unique mechanism and step-like characteristics with well-defined discreet openings and closings. The molecule was incorporated into the membrane in a reproducible way, and the observed channel-like activity could be responsible for the antibacterial activity of this molecule.

Application of Machine-Learning Methods to Recognize mitoBK Channels from Different Cell Types Based on the Experimental Patch-Clamp Results.

(1) Background: In this work, we focus on the activity of large-conductance voltage- and Ca2+-activated potassium channels (BK) from the inner mitochondrial membrane (mitoBK). The characteristic electrophysiological features of the mitoBK channels are relatively high single-channel conductance (ca. 300 pS) and types of activating and deactivating stimuli. Nevertheless, depending on the isoformal composition of mitoBK channels in a given membrane patch and the type of auxiliary regulatory subunits (which can be co-assembled to the mitoBK channel protein) the characteristics of conformational dynamics of the channel protein can be altered. Consequently, the individual features of experimental series describing single-channel activity obtained by patch-clamp method can also vary. (2) Methods: Artificial intelligence approaches (deep learning) were used to classify the patch-clamp outputs of mitoBK activity from different cell types. (3) Results: Application of the K-nearest neighbors algorithm (KNN) and the autoencoder neural network allowed to perform the classification of the electrophysiological signals with a very good accuracy, which indicates that the conformational dynamics of the analyzed mitoBK channels from different cell types significantly differs. (4) Conclusion: We displayed the utility of machine-learning methodology in the research of ion channel gating, even in cases when the behavior of very similar microbiosystems is analyzed. A short excerpt from the patch-clamp recording can serve as a "fingerprint" used to recognize the mitoBK gating dynamics in the patches of membrane from different cell types.

Functional Expression of TRPV1 Ion Channel in the Canine Peripheral Blood Mononuclear Cells.

TRPV1, known as a capsaicin receptor, is the best-described transient receptor potential (TRP) ion channel. Recently, it was shown to be expressed by non-excitable cells such as lymphocytes. However, the data regarding the functional expression of the TRPV1 channel in the immune cells are often contradictory. In the present study, we performed a phylogenetical analysis of the canine TRP ion channels, we assessed the expression of TRPV1 in the canine peripheral blood mononuclear cells (PBMC) by qPCR and Western blot, and we determined the functionality of TRPV1 by whole-cell patch-clamp recordings and calcium assay. We found high expression of TRPV2, -M2, and -M7 in the canine PBMCs, while expression of TRPV1, -V4 and, -M5 was relatively low. We confirmed that TRPV1 is expressed on the protein level in the PBMC and it localizes in the plasma membrane. The whole-cell patch-clamp recording revealed that capsaicin application caused a significant increase in the current density. Similarly, the results from the calcium assay show a dose-dependent increase in intracellular calcium level in the presence of capsaicin that was partially abolished by capsazepine. Our study confirms the expression of TRPV1 ion channel on both mRNA and protein levels in the canine PBMC and indicates that the ion channel is functional.

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 ß3-ß4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately ß3 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.

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 (TNF-) 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.

Naringenin as an opener of mitochondrial potassium channels in dermal fibroblasts.

Flavonoids belong to a large group of polyphenolic compounds that are widely present in plants. Certain flavonoids, including naringenin, have cytoprotective properties. Although the antioxidant effect has long been thought to be a crucial factor accounting for the cellular effects of flavonoids, mitochondrial channels have emerged recently as targets for cytoprotective strategies. In the present study, we characterized interactions between naringenin and the mitochondrial potassium (mitoBKCa and mitoKATP ) channels recently described in dermal fibroblasts. With the use of the patch-clamp technique and mitoplasts isolated from primary human dermal fibroblast cells, our study shows that naringenin in micromolar concentrations leads to an increase in mitoBKCa channel activity. The opening probability of the channel decreased from 0.97 in the control conditions (200 µmol/L Ca2+ ) to 0.06 at a low Ca2+ level (1 µmol/L) and increased to 0.85 after the application of 10 µmol/L naringenin. Additionally, the activity of the mitoKATP channel increased following the application of 10 µmol/L naringenin. To investigate the effects of naringenin on mitochondrial function, the oxygen consumption of dermal fibroblast cells was measured in potassium-containing media. The addition of naringenin significantly and dose-dependently increased the respiratory rate from 5.8 ± 0.2 to 14.0 ± 0.6 nmol O2  × min-1  × mg protein-1 . Additionally, a Raman spectroscopy analysis of skin penetration indicated that the naringenin was distributed in all skin layers, including the epidermis and dermis. In this study, we demonstrated that a flavonoid, naringenin, can activate two potassium channels present in the inner mitochondrial membrane of dermal fibroblasts.

Single-Channel Properties of the ROMK-Pore-Forming Subunit of the Mitochondrial ATP-Sensitive Potassium Channel.

An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.

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.

cGMP-Elevating Compounds and Ischemic Conditioning Provide Cardioprotection Against Ischemia and Reperfusion Injury via Cardiomyocyte-Specific BK Channels.

BACKGROUND: The nitric oxide-sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca2+-activated K+ channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types. METHODS: Gene-targeted mice with a cardiomyocyte- or smooth muscle cell-specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model. RESULTS: Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide-sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice. CONCLUSIONS: Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.

What do we not know about mitochondrial potassium channels?

In this review, we summarize our knowledge about mitochondrial potassium channels, with a special focus on unanswered questions in this field. The following potassium channels have been well described in the inner mitochondrial membrane: ATP-regulated potassium channel, Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the two-pore domain TASK-3 potassium channel. The primary functional roles of these channels include regulation of mitochondrial respiration and the alteration of membrane potential. Additionally, they modulate the mitochondrial matrix volume and the synthesis of reactive oxygen species by mitochondria. Mitochondrial potassium channels are believed to contribute to cytoprotection and cell death. In this paper, we discuss fundamental issues concerning mitochondrial potassium channels: their molecular identity, channel pharmacology and functional properties. Attention will be given to the current problems present in our understanding of the nature of mitochondrial potassium channels. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria.

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and ß-subunits (predominantly the ß3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100µM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

[What we don't know about mitochondrial potassium channels?] / Czego nie wiemy o mitochondrialnych kanalach potasowych?

In the current work the authors present the most interesting, yet not fully understood issues regarding origin, function and pharmacology of the mitochondrial potassium channels. There are eight potassium channels known to contribute to the potassium permeability of the inner mitochondrial membrane: ATP-regulated channel, calcium-regulated channels of large, intermediate and small conductance, voltage-regulated Kv1.3 and Kv7.4 channels, two-pore-domain TASK-3 channel and SLO2 channel. The primary function of the mitochondrial potassium channels is regulation of the mitochondrial membrane potential. Additionally, mitochondrial potassium channels alter cellular respiration, regulation of the mitochondrial volume and ROS synthesis. However, mechanisms underlying these processes are not fully understood yet. In this work, the authors not only present available knowledge about this topic, but also put certain hypotheses that may set the direction for the future research on these proteins.