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Douglas R. Pfeiffer - One of the best experts on this subject based on the ideXlab platform.

  • regulation of the mitochondrial ca2 uniporter by external adenine nucleotides the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations
    Biochemistry, 1997
    Co-Authors: Monica L Litsky, Douglas R. Pfeiffer
    Abstract:

    We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+ uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+ is reversed by spermine (EC0.5 ≈ 40 μM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP ≈ UTP > GTP. The effectiveness of ATP (EC50 ≈ 0.6 mM) is the same in mitochondria and Mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxy...

  • regulation of the mitochondrial ca2 uniporter by external adenine nucleotides the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations
    Biochemistry, 1997
    Co-Authors: Monica L Litsky, Douglas R. Pfeiffer
    Abstract:

    We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+ uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+ is reversed by spermine (EC0.5 approximately 40 microM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP approximately UTP > GTP. The effectiveness of ATP (EC50 approximately 0.6 mM) is the same in mitochondria and Mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxyatractyloside, or AP5A, used alone or in combinations. These findings indicate that ATP acts at a site located on the outer surface of the inner membrane through a mechanism which does not require its hydrolysis. Phosphate also inhibits reverse uniport under some conditions (EC50 approximately 20 microM). The sites at which free ATP and free Mg2+ inhibit the uniporter can be distinguished by chymotrypsin treatment of Mitoplasts, which eliminates the action of Mg2+ but does not affect the action of ATP. Data are interpreted within the context of a model in which the uniporter is considered to be a gated channel that is controlled, in part, by specific external effector sites that accept divalent cations or nucleotides. The possible consequences of the model for cell Ca2+ regulation by mitochondria and regulation of TCA cycle activity by the matrix free Ca2+ concentration are considered.

Jose C Fernandezcheca - One of the best experts on this subject based on the ideXlab platform.

  • cholesterol impairs the adenine nucleotide translocator mediated mitochondrial permeability transition through altered membrane fluidity
    Journal of Biological Chemistry, 2003
    Co-Authors: Anna Colell, Carmen Garciaruiz, Josep M Lluis, Olga Coll, Montse Mari, Jose C Fernandezcheca
    Abstract:

    Abstract Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.

  • tauroursodeoxycholic acid protects hepatocytes from ethanol fed rats against tumor necrosis factor induced cell death by replenishing mitochondrial glutathione
    Hepatology, 2001
    Co-Authors: Anna Colell, Carmen Garciaruiz, Olga Coll, Raquel Paris, Claudio Tiribelli, Neil Kaplowitz, Jose C Fernandezcheca
    Abstract:

    Abstract Mitochondrial glutathione (GSH) plays a key role against tumor necrosis factor α (TNF)-induced apoptosis because its depletion is known to sensitize hepatocytes to TNF. The present study examined the role of tauroursodeoxycholic acid (TUDCA) administration to chronic ethanol-fed rats on mitochondrial GSH levels and kinetics, mitochondrial membrane physical properties, TNF-induced peroxide formation, and subsequent hepatocyte survival. TUDCA selectively increased the levels of GSH in mitochondria without an effect on cytosolic GSH. This outcome was accompanied by improved initial rate of GSH transport examined at low (1 mmol/L) and high (10 mmol/L) GSH concentrations both in intact mitochondria and Mitoplasts prepared from ethanol-fed livers. Assessment of membrane fluidity revealed an increased order parameter in mitochondria and Mitoplasts from ethanol-fed rats compared with pair-fed controls, which was prevented by TUDCA administration. Compared with hepatocytes from pair-fed rats, TNF stimulated peroxide generation in hepatocytes from ethanol-fed rats, preceding TNF-induced cell death. Administration of TUDCA to ethanol-fed rats prevented TNF-induced peroxide formation and cell death, an effect that was reversed on depletion of the recovered mitochondrial GSH levels by (R,S)-3-hydroxy-4-pentenoate before TNF treatment. The protective effect of TUDCA against TNF was not because of activation of phosphatidylinositol 3-kinase, discarding a role for a survival-dependent pathway. Thus, these findings reveal a novel role of TUDCA in protecting hepatocytes in long-term ethanol-fed rats through modulation of mitochondrial membrane fluidity and subsequent normalization of mitochondrial GSH levels. (H EPATOLOGY 2001;34:964-971.)

Maria Barile - One of the best experts on this subject based on the ideXlab platform.

  • the purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds fad via an autocatalytic reaction
    International Journal of Biological Macromolecules, 2008
    Co-Authors: Carmen Brizio, Roderich Brandsch, Maria Douka, Robin Wait, Maria Barile
    Abstract:

    Abstract The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me 2 GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver Mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me 2 GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.

  • a protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase
    FEBS Journal, 2000
    Co-Authors: Carmen Brizio, Annegret Otto, Roderich Brandsch, Salvatore Passarella, Maria Barile
    Abstract:

    The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized Mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized Mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.

Detlef Siemen - One of the best experts on this subject based on the ideXlab platform.

  • direct inhibition of the mitochondrial permeability transition pore a possible mechanism for better neuroprotective effects of allopregnanolone over progesterone
    Brain Research, 2009
    Co-Authors: Iqbal Sayeed, Detlef Siemen, Suhel Parvez, Bushra Wali, Donald G Stein
    Abstract:

    We previously demonstrated that the progesterone (PROG) metabolite allopregnanolone (AP) is more potent than PROG in the treatment of traumatic brain injury (TBI) and stroke, but the mechanisms for this differential effect are little understood. The mitochondrial permeability transition pore (mtPTP) appears to be a key player in the intrinsic pathway of apoptosis-induced loss of neurons. Its activation is accompanied by the release of cytochrome c (cyt c) from the intermembrane gap and subsequent cell death. We investigated whether mtPTP is implicated in the mechanisms of PROG and AP neuroprotection following traumatic and ischemic brain injury. To assess the neurosteroids' direct effects on mtPTP activity at the single-channel level, recordings from the inner mitochondrial membrane were obtained by a patch-clamp approach in rat liver Mitoplasts. AP but not PROG strongly inhibited mtPTP currents. Interaction of AP with the PTP was further supported by a swelling assay demonstrating that AP inhibited Ca(2+)-triggered swelling in functionally intact rat liver and brain mitochondria. If AP inhibits the mtPTP, it should prevent the mitochondrial cyt c release seen in stroke and TBI. To test this idea, we subjected one group of rats to cortical contusion injury (CCI) and another to transient middle cerebral artery occlusion (MCAO). AP-treated animals showed substantially decreased cyt c release and AP was more potent than PROG in inhibiting mitochondrial cyt c release at 24 h post-CCI and -MCAO. Our results demonstrate that AP inhibits the mtPTP current. This may help to explain its more potent anti-apoptotic and neuroprotective effects compared to PROG.

  • hypoxia increases activity of the bk channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore
    Cellular Physiology and Biochemistry, 2008
    Co-Authors: Yu Cheng, Piotr Bednarczyk, Falk R Wiedemann, Gabriel G Haddad, Detlef Siemen
    Abstract:

    Hypoxia can cause severe damage to cells by initiating signaling cascades that lead to cell death. A cellular oxygen sensor, other than the respiratory chain, might exist in sensitive components of these signaling cascades. Recently, we found evidence that mitochondrial ion channels are sensitive to low levels of oxygen. We therefore studied the effects of hypoxia on the mitochondrial BK-channel (mtBK), on the mitochondrial permeability transition pore (PTP), and on their possible interaction. Using single-channel patch-clamp techniques we found that hypoxia inhibited the PTP but substantially increased the mtBK activity of Mitoplasts from rat liver and astrocytes. Experiments measuring the mitochondrial membrane potential of intact rat brain mitochondria (using the fluorescence dye safranine O) during hypoxia exhibited an increased Ca(2+)-retention capacity implying an impaired opening of the PTP. We also found a reduced Ca(2+)-retention capacity with 100 nM iberiotoxin, a selective inhibitor of BK-channels. We therefore conclude that there is interaction between the mtBK and the PTP in a way that an open mtBK keeps the PTP closed. Thus, the response of mitochondrial ion channels to hypoxia could be interpreted as anti-apoptotic.

  • the human mitochondrial katp channel is modulated by calcium and nitric oxide a patch clamp approach
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Yuliya A Dahlem, Thomas F W Horn, Linas Buntinas, Tohru Gonoi, Gerald Wolf, Detlef Siemen
    Abstract:

    ATP-sensitive potassium channels of the inner mitochondrial membrane (mtKATP) are blocked by ATP. They are suggested to be involved in protective mechanisms such as ischemic preconditioning (IPC). Here we identify this channel type for the first time in a human cell line (Jurkat cells). Vesicles of the inner mitochondrial membrane (Mitoplasts) were prepared by hypoosmotic shock. Single-channel currents were measured by means of the patch-clamp technique. We identified an outward-rectifying channel with a slope conductance of 15 and 82 pS at negative and positive potentials, respectively. The block by 5-hydroxydecanoic acid and inhibition by ATP characterize this channel as the mtKATP channel. ATP also increased the frequency of events within the burst. This effect was modulated by the Ca2+-bath concentration. We also show that the human mtKATP channel is a direct target for nitric oxide that blocked the channel activity. Although the molecular structure of this channel type is still unknown, its characterization as an outward-rectifying channel and modulation by calcium ions and nitric oxide may help to elucidate its functional significance, which possibly implicates a role in cell survival after IPC.

  • ca2 activated k channel of the bk type in the inner mitochondrial membrane of a human glioma cell line
    Biochemical and Biophysical Research Communications, 1999
    Co-Authors: Detlef Siemen, Christos Loupatatzis, Jiri Borecky, Erich Gulbins, Florian Lang
    Abstract:

    A single channel current was recorded from Mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the Mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the Mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.

Adam Szewczyk - One of the best experts on this subject based on the ideXlab platform.

  • single channel properties of the romk pore forming subunit of the mitochondrial atp sensitive potassium channel
    International Journal of Molecular Sciences, 2019
    Co-Authors: Michal Laskowski, Monika żochowska, Justyna Kalisz, Bartlomiej Augustynek, Brian Orourke, Adam Szewczyk, Piotr Bednarczyk, Bogusz Kulawiak
    Abstract:

    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.

  • Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties
    MDPI AG, 2018
    Co-Authors: Bartlomiej Augustynek, Adam Szewczyk, Piotr Koprowski, Daria Rotko, Wolfram S. Kunz, Bogusz Kulawiak
    Abstract:

    Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma Mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models

  • Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures.
    Acta Neurobiologiae Experimentalis, 2016
    Co-Authors: Marta Piwońska, Adam Szewczyk, Ulrich H. Schröder, Klaus G. Reymann, Piotr Bednarczyk
    Abstract:

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

  • CALL FOR PAPERS Mitochondria in Cardiovascular Physiology and Disease Large-conductance Ca2-activated potassium channel in mitochondria of endothelial EA.hy926 cells
    2013
    Co-Authors: Piotr Bednarczyk, Agnieszka Koziel, Wieslawa Jarmuszkiewicz, Adam Szewczyk
    Abstract:

    Physiol 304: H1415–H1427, 2013. First published March 29, 2013; doi:10.1152/ajpheart.00976.2012.—In the present study, we describe the existence of a large-conductance Ca2-activated potas-sium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial Mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the Mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irrevers-ibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced th

  • A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.
    Biochimica et biophysica acta, 2012
    Co-Authors: Anna Kajma, Adam Szewczyk
    Abstract:

    Abstract Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288 ± 4 pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150 mM KCl). At positive voltages, this channel displayed a conductance of 67.84 pS and a strong voltage dependence at holding potentials from − 80 mV to + 80 mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the Mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).