Regulation Of Permeability Transition Pore Research Articles

Effects of Decreasing Mitochondrial Volume on the Regulation of the Permeability Transition Pore

The permeability transition pore (PTP) is a Ca(2+)-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca(2+) threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg(2+). By studying the effects of Mg(2+), cyclosporin A (CsA) and ubiquinone 0 (Ub(0)) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg(2+) and Ub(0) at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

Mitochondrial permeability transition-pore inhibition enhances functional recovery after long-time hypothermic heart preservation.

Long-time preservation of the donor heart before transplantation is associated with mitochondrial damages resulting in functional deterioration after transplantation, although the exact mechanism behind this is still uncertain. Here, we have demonstrated the opening of a nonspecific pore in the inner membrane of the mitochondria, the mitochondrial permeability transition pore (PTP), as one of the reasons responsible for this functional deterioration. After 30 minutes of perfusion with the Krebs Henseleit buffer at 37 degrees C in working mode, hearts from Wistar rats were arrested with ice-cold St. Thomas cardioplegic solution and preserved University of Wisconsin solution at 4 degrees C with or without inhibiting the PTP with cyclosporin A (CsA) (0.2 microM). After 12 hours, the hearts were reperfused for 60 minutes at 37 degrees C with or without perfusing the hearts during first 15 minutes of reperfusion with PTP openers lonidamine (30 microM) or atractyloside (20 microM). Inhibiting PTP with CsA resulted in (a) significant recovery of cardiac functions, (b) well-preserved myocardial adenosine triphosphate (ATP) levels (P<0.001), (c) less myocardial water content (P<0.01), (d) less mitochondrial swelling and cytochrome C release, and (e) up-regulation of Bcl-2 expression compared with the control hearts without PTP inhibition. These effects are completely inhibited on opening the PTP before preservation, resulting in poor recovery of cardiac functions, loss of myocardial ATP, and severe mitochondrial swelling. The present study demonstrates the potential role of mitochondrial PTP after long- time hypothermic preservation of the heart, and therefore, regulation of PTP would prolong the preservation time of donor hearts.

Role of substrates in the regulation of mitochondrial function in situ.

Investigations of mitochondrial oxidative phosphorylation have been mainly carried out in isolated mitochondria, where the experimental conditions can be precisely set. However, in intact living systems oxidative phosphorylation takes place in a complex environment, whose experimental dissection is a major challenge. It has long been recognized that the efficiency of oxidative phosphorylation depends on the nature of the respiratory substrates, which feed electrons to the respiratory chain at different levels. Yet, the role of substrates in determining mitochondrial function and their response to energetic stress has been largely overlooked. Here we review recent work showing that the nature of the energetic substrates profoundly affects the mitochondrial responses to manipulations of pathophysiological relevance, such as uncoupling and opening of the permeability transition pore (PTP). Uncoupling of intact hepatocytes caused very different metabolic effects depending on whether carbohydrates or lipids were the energy source. With dihydroxyacetone as the substrate dinitrophenol caused a collapse of the mitochondrial membrane potential and of the ATP/ADP ratio, while the respiratory rate was increased only transiently. With octanoate as the substrate, on the other hand, dinitrophenol caused a dramatic stimulation of the respiratory rate, while the mitochondrial membrane potential and ATP/ADP ratio were affected only marginally. We then review results indicating that the activity of complex I directly regulates the PTP, a finding that emphasizes the importance of the respiratory substrates in PTP regulation.

Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells.

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.

Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function

Mitochondria play a central role in energy metabolism, Ca(2+) signalling, aging and cell death. To control cytosolic or mitochondrial Ca(2+) concentration, mitochondria possess several Ca(2+)-transport systems across the inner membrane. However, the pathway for Ca(2+) crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca(2+). VDAC contains Ca(2+)-binding sites that bind Ruthenium Red (RuR), La(3+) and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca(2+) (500-700 nmol/mg of protein), and subsequently released it. The release of Ca(2+) is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca(2+) accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca(2+) and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca(2+) transport into and from the mitochondria, and that the inhibition of Ca(2+) uptake by RuR and La(3+) may result from their interaction with VDAC Ca(2+)-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca(2+) and its binding of Ca(2+), suggest that VDAC has a role in regulation of the mitochondrial Ca(2+) homoeostasis.

Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function

Mitochondria play a central role in energy metabolism, Ca2+ signalling, aging and cell death. To control cytosolic or mitochondrial Ca2+ concentration, mitochondria possess several Ca2+-transport systems across the inner membrane. However, the pathway for Ca2+ crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca2+. VDAC contains Ca2+-binding sites that bind Ruthenium Red (RuR), La3+ and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca2+ (500–700nmol/mg of protein), and subsequently released it. The release of Ca2+ is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca2+ accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca2+ and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca2+ transport into and from the mitochondria, and that the inhibition of Ca2+ uptake by RuR and La3+ may result from their interaction with VDAC Ca2+-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca2+ and its binding of Ca2+, suggest that VDAC has a role in regulation of the mitochondrial Ca2+ homoeostasis.

A Ubiquinone-binding Site Regulates the Mitochondrial Permeability Transition Pore

We have investigated the regulation of the mitochondrial permeability transition pore (PTP) by ubiquinone analogues. We found that the Ca2+-dependent PTP opening was inhibited by ubiquinone 0 and decylubiquinone, whereas all other tested quinones (ubiquinone 5, 1,4-benzoquinone, 2-methoxy-1,4-benzoquinone, 2,3-dimethoxy-1, 4-benzoquinone, and 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone) were ineffective. Pore inhibition was observed irrespective of the method used to induce the permeability transition (addition of Pi or atractylate, membrane depolarization, or dithiol cross-linking). Inhibition of PTP opening by decylubiquinone was comparable with that exerted by cyclosporin A, whereas ubiquinone 0 was more potent. Ubiquinone 5, which did not inhibit the PTP per se, specifically counteracted the inhibitory effect of ubiquinone 0 or decylubiquinone but not that of cyclosporin A. These findings define a ubiquinone-binding site directly involved in PTP regulation and indicate that different quinone structural features are required for binding and for stabilizing the pore in the closed conformation. At variance from all other quinones tested, decylubiquinone did not inhibit respiration. Our results define a new structural class of pore inhibitors and may open new perspectives for the pharmacological modulation of the PTP in vivo.

Microtubule-active drugs suppress the closure of the permeability transition pore in tumour mitochondria

We report the effects of anticancer drugs, inhibitors of microtubule organisation, on the mitochondrial permeability transition pore (PTP) in Ehrlich ascites tumour cells. Taxol (5–20 μM) and colchicine (100–500 μM) prevented closing of the cyclosporin A-sensitive PTP. No taxol or colchicine effects on oxidative phosphorylation were observed in the range of concentrations used. We suggest that either membrane-bound tubulin per se can be part of PTP and/or the attachment of mitochondria to the microtubular network is essential for PTP regulation. The taxol inhibition of PTP closure, mediated through interaction with the cytoskeleton, sheds new light on the cytotoxic properties of this anticancer drug.