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Neutral rhodol-based red emitters are shown to efficiently localize in mitochondria, as demonstrated by confocal microscopy and co-localization studies. A simple model is proposed to explain the localization mechanism of neutral molecules. The model takes into account the strong coupling between the molecular dipole moment and the electric field of the inner mitochondrial membrane.
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Colorantes Fluorescentes , Mitocondrias , Mitocondrias/metabolismo , Mitocondrias/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Humanos , Microscopía Confocal , Xantonas/química , Estructura Molecular , Células HeLaRESUMEN
In 2024, it will be 140 years since the birth of Jan Karol Parnas, one of the most outstanding biochemists and one of the most outstanding Polish scientists of the first half of the 20th century. The article presents a synthetic biography of Prof. Parnas, his influence on the organization of scientific life in interwar Poland, and presents his students and collaborators who created the so-called "The School of Parnassus". It shows how this figure still influences the thinking of Polish biochemists about their Society and the attitude towards biochemical communities abroad, especially in Ukraine, which owes as much to Prof. Parnas as Poland does.
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Bioquímica , Polonia , Historia del Siglo XX , Bioquímica/historia , Aniversarios y Eventos Especiales , Historia del Siglo XXI , UcraniaRESUMEN
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.
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Glioblastoma , Canales de Potasio de Gran Conductancia Activados por el Calcio , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Glioblastoma/metabolismo , Mitocondrias/metabolismo , Potasio/metabolismo , Calcio/metabolismoRESUMEN
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.
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Sulfuro de Hidrógeno , Canales de Potasio de Gran Conductancia Activados por el Calcio , Sitios de Unión , Calcio/metabolismo , Hemo/metabolismo , Hemina/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , Hierro/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismoRESUMEN
Novel highly sensitive fluorescent probes for zinc cations based on the diketopyrrolopyrrole scaffold were designed and synthesized. Large bathochromic shifts (≈80 nm) of fluorescence are observed when the Zn2+-recognition unit (di-(2-picolyl)amine) is bridged with the fluorophore possessing an additional pyridine unit able to participate in the coordination process. This effect originates from the dipolar architecture and the increasing electron-withdrawing properties of the diketopyrrolopyrrole core upon addition of the cation. The new, greenish-yellow emitting probes, which operate via modulation of intramolecular charge transfer, are very sensitive to the presence of Zn2+. Introduction of a morpholine unit in the diketopyrrolopyrrole structure induces a selective six-fold increase of the emission intensity upon zinc coordination. Importantly, the presence of other divalent biologically relevant metal cations has negligible effects and typically even at a 100-fold higher concentration of Mg2+/Zn2+, the effect is comparable. Computational studies rationalize the strong bathochromic shift upon Zn2+-complexation. Decorating the probes with the triphenylphosphonium cation and morpholine unit enables selective localization in the mitochondria and the lysosome of cardiac H9C2 cells, respectively.
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Colorantes Fluorescentes , Zinc , Aminas , Cationes Bivalentes , Colorantes Fluorescentes/química , Cetonas , Morfolinas , Piridinas , Pirroles , Espectrometría de Fluorescencia , Zinc/químicaRESUMEN
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.
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Calcitriol , Canales de Potasio de Gran Conductancia Activados por el Calcio , Calcitriol/metabolismo , Calcitriol/farmacología , Calcio/metabolismo , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/farmacología , Mitocondrias/metabolismo , Simulación del Acoplamiento Molecular , Técnicas de Placa-ClampRESUMEN
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.
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Calcio , Hemina , Hemina/farmacología , Hemina/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismoRESUMEN
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.
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Mitocondrias/metabolismo , Modelos Biológicos , Canales de Potasio/análisis , Canales de Potasio/metabolismo , Animales , Humanos , Transporte IónicoRESUMEN
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.
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Antibacterianos/química , Membrana Dobles de Lípidos/química , Polímeros/química , Antibacterianos/metabolismo , Concentración de Iones de Hidrógeno , Iones/química , Membrana Dobles de Lípidos/metabolismo , Magnesio/química , Permeabilidad , Polietileneimina/química , Polímeros/síntesis química , Polímeros/metabolismoRESUMEN
In this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochrome c oxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochrome c oxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized. Interestingly, supercomplexes of COX formed with complex III and also with complexes I and III retained considerable immunoreactivity, while nearly no immunoreactivity attributable to subassemblies was found. That indicates that COX lacking subunit 8A is stabilized in supercomplexes, while monomers and dimers are rapidly degraded. With transcriptome analysis by 3'-RNA sequencing we failed to detect in our cellular models of COX8A deficiency transcriptional changes of genes involved in the mitochondrial unfolded protein response (mtUPR) and the integrated stress response (ISR). Thus, our data strongly suggest that the smallest subunit of cytochrome c oxidase COX8A is required for maintenance of the structural stability of COX monomers and dimers.
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Transporte de Electrón/genética , Mitocondrias/enzimología , Mutación , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
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.
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Bronquios/metabolismo , Calcio/metabolismo , Células Epiteliales/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Consumo de Oxígeno , Potasio/metabolismo , Biofisica , Supervivencia Celular , Electrofisiología , Metabolismo Energético , Epitelio/metabolismo , Humanos , Indoles/química , Potencial de la Membrana Mitocondrial , Potenciales de la Membrana , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Técnicas de Placa-Clamp , Péptidos/química , Dominios ProteicosRESUMEN
The sudden death of Professor Lech Wojtczak, the great Polish biochemist and a remarkable man, our Mentor and Friend, left us in sorrow and emptiness difficult to accept. Two years have passed already from this event and our memories seem to be even more vivid, and his absence even more felt. Hence we decided to put on paper our personal reflections on Lech Wojtczak, each of us concentrating on a slightly different aspect of this towering figure. We tried to focus on memories and comments that were not mentioned in official obituaries that followed His passing away. Therefore do not expect to find here a comprehensive text on the Founder of Polish Bioenergetics, and a famous Polish biochemist, but rather a set of subjective comments on a man who made us scientists. Our memories are presented in a chronological order. The first chapter is by Professor Jolanta Baranska, who joined the group of Lech Wojtczak in 1968, followed by a chapter by Professor Maciej J. Nalecz, who joined Lech in 1976, then Professor Konrad S. Famulski (1978) and finally followed by a chapter by Professor Adam Szewczyk, the youngest, joining the group in 1984.
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Metabolismo Energético , Estudiantes , Humanos , Masculino , PoloniaRESUMEN
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.
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Citrus/química , Endotelio Vascular/efectos de los fármacos , Flavanonas/farmacología , Flavonoides/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Mitocondrias/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Citoprotección , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Transporte Iónico , Potenciales de la MembranaRESUMEN
Relacja z "8th MITOCHONDRION: in memory of Professor Lech Wojtczak".
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Mitocondrias , Historia del Siglo XXRESUMEN
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.
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Fibroblastos/efectos de los fármacos , Flavanonas/farmacología , Canales de Potasio/metabolismo , Piel/efectos de los fármacos , Adulto , Antioxidantes/metabolismo , Mama/metabolismo , Calcio/metabolismo , Células Cultivadas , Dermis/metabolismo , Diazóxido/farmacología , Femenino , Fibroblastos/citología , Humanos , Mitocondrias/metabolismo , Oxígeno/metabolismo , Consumo de Oxígeno , Técnicas de Placa-Clamp , Piel/citología , Espectrometría RamanRESUMEN
Energy homeostasis in mitochondria is vital for proper muscle cell function. In this review we will focus on cardiac and skeletal muscle energy-dissipating systems, i.e., mitochondrial potassium channels and uncoupling proteins. Despite the molecular differences between these proteins both of them may regulate the generation of reactive oxygen species. Hence, they can both modulate pro-life and -death signaling in response to the needs of the muscle cell. Certain mitochondrial potassium channels (such as the ATP-regulated and large conductance calcium-activated mitochondrial potassium channels) and uncoupling proteins may be regulated in a similar manner suggesting that both are part of the energy-dissipating hub in muscle mitochondria. Understanding the role of these proteins, especially in the context of ischemia-reperfusion injury of cardiac muscle, may be important for pharmacological intervention. This review highlights several aspects of the regulation of mitochondrial potassium channels and uncoupling proteins in muscle mitochondria and their association with diseases.
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Metabolismo Energético , Mitocondrias Musculares/metabolismo , Proteínas Desacopladoras Mitocondriales/metabolismo , Canales de Potasio/metabolismo , Animales , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
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Adenosina Trifosfato/metabolismo , Magnesio/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Adenosina Trifosfato/genética , Línea Celular , Humanos , Proteínas Mitocondriales/genética , Canales de Potasio de Rectificación Interna/genéticaRESUMEN
Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBKCa) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBKCa channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBKCa channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BKCa-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BKCa splice variants that variable mechanosensitivity of mitoBKCa could be the result of the presence of diverse BKCa isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBKCa channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.
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Calcio/metabolismo , Glioma/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Mecanotransducción Celular , Mitocondrias/metabolismo , Glioma/patología , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potencial de la Membrana Mitocondrial , Mutación , Técnicas de Placa-Clamp , Células Tumorales CultivadasRESUMEN
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 150â¯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.
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Dermis/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Línea Celular , Dermis/citología , Fibroblastos/citología , Humanos , Mitocondrias/genética , Canales de Potasio de Rectificación Interna/genéticaRESUMEN
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.