Mitochondrial ADP serves at least two important functions: provides a substrate for ATP generation and inhibits the mitochondrial permeability transition pore (mPTP). The mechanism for the inhibitory effect of ADP on mPTP remains unclear. Moreover, the contribution of this ADP-dependent regulation of mPTP in protection from mitochondrial injury has yet to be elucidated. We investigated the inhibitory effects of ADP on mPTP in mitochondria isolated from mouse hearts by measuring mitochondrial Ca2+ uptake capacity.

AbstractOriginally published in: Bacterial and Eukaryotic Porins. Edited by Roland Benz. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30775‐3The sections in this article areIntroductionStructure and Isotypes of VDACThe Influence of Phospholipids on VDAC StructureVDAC Conductance and Ion SelectivityPhysiological Significance of the Voltage DependencePorins as Specific Binding SitesVDAC senses Inner Membrane Functions in the Contact SiteCytochromecis a Component of the Contact SitesIsolation and Characterization of VDAC‐ANT ComplexesReconstitution of VDAC‐ANT ComplexesImportance of Metabolic Channeling in Regulation of Energy MetabolismThe VDAC‐ANT Complex as Permeability Transition PoreThe VDAC‐ANT Complex as a Target for Bax‐dependent CytochromecReleaseThe VDAC–ANT Complexes contain CytochromecThe Importance of the Kinases in Regulation of ApoptosisSuppression of Bax‐dependent CytochromecRelease and Permeability Transition by HexokinaseSuppression of Permeability Transition by Mitochondrial Creatine KinaseConclusion

The permeability transition pore (PT-pore) is a multi-component protein aggregate in mitochondria that comprises factors in the inner as well as in the outer mitochondrial membrane. This complex has two functions: firstly, it regulates the integration of oxidative phosphorylation into the cellular energy household and secondly, it induces cell death when converted into an unspecific channel. The latter causes a collapse of the mitochondrial membrane potential and activates a chain of events that culminate in the demise of the cell. It has been controversial for some time whether the PT-pore is causative for or only amplifies a signal of cell death but novel results confirm a central role of this protein complex for cell death induction. While a considerable body of data exist on its subunit composition, recent genetic knock-out experiments suggest that the identity of the core factors of the PT-pore is still unresolved. Moreover, accumulating evidence point to a much more complex composition of this protein complex than anticipated. Here, we review the current knowledge of its subunit composition, the evidence of a role in cell death, and we propose a model for the activation of the PT-pore for cell death.

This chapter contains sections titled: Abstract The Role of VDAC in Controlling the Interaction of Mitochondria with the Cytosol Molecular Structure and Membrane Topology of VDAC VDAC Conductance, Voltage Dependence, and Ion Selectivity The Physiological Significance of VDAC Voltage Gating VDAC Isoforms and Functions Mitochondria and Apoptosis VDAC and ANT May Form the Mitochondrial Permeability Transition Pore Accessory MPT Pore Subunits ANT Knockout Studies The Role of VDAC in Organizing Kinases at the Mitochondrial Surface Hexokinase–VDAC–ANT Complexes in Tumor Cells Hexokinase as a Marker Enzyme of Contact Sites VDAC–ANT Complexes VDAC–ANT Complexes Contain Cytochrome c VDAC Oligomerization and Cytochrome c Binding Possible Function of Cytochrome c in the Contact Sites Cholesterol and Cardiolipin Influence VDAC Structure and Function The Importance of VDAC Complexes in Regulation of Energy Metabolism and Apoptosis Suppression of Bax-dependent Cytochrome c Release and Permeability Transition by Hexokinase Suppression of Permeability Transition and Cytochrome c Release by Mitochondrial Creatine Kinase The General Importance of the Creatine Kinase System in Heart Performance The Central Regulatory Role of ANT References

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

The energy metabolism of the failing heart is characterised by a 30% decrease of the total adenine nucleotides content and what may be more important by a 60% loss of creatine and creatine phosphate [J.S. Ingwall, R.G. Weiss, Is the failing heart energy starved? On using chemical energy to support cardiac function, Circ. Res. 95 (2004) 35-145]. Besides the effect of these changes on the energy supply, failing heart is known to be more vulnerable to Ca2+ overload and apoptosis-inducing processes. Recent studies have pointed to the critical role of mitochondrial contact sites in controlling both the mitochondrial energy metabolism and Ca2+ homeostasis. This review focuses on the structure and function of protein complexes in mitochondrial contact sites and their regulatory role in the cellular bioenergetics, intra- and extra-mitochondrial Ca2+ levels, and release of apoptosis-inducing factors. Firstly, we review the compositions of different contact sites following by the discussion of experimental data obtained with isolated and reconstituted voltage-dependent anion channel-adenine nucleotide translocase complexes and consequences of the complex disassembling. Furthermore, we describe experiments involving the complex-stabilizing conditions in vitro and in intact cells. At the end, we discuss unsolved problems and opportunities for clinical application of the complex-stabilizing factors.

We have investigated the role of the protein ubiquitous mitochondrial creatine kinase (uMtCK) in the formation and stabilization of inner and outer membrane contact sites. Using liver mitochondria isolated from transgenic mice, which, unlike control animals, express uMtCK in the liver, we found that the enzyme was associated with the mitochondrial membranes and, in addition, was located in membrane-coated matrix inclusions. In mitochondria isolated from uMtCK transgenic mice, the number of contact sites increased 3-fold compared with that observed in control mitochondria. Furthermore, uMtCK-containing mitochondria were more resistant to detergent-induced lysis than wild-type mitochondria. We conclude that octameric uMtCK induces the formation of mitochondrial contact sites, leading to membrane cross-linking and to an increased stability of the mitochondrial membrane architecture.

The pro-apoptotic protein Bax plays a key role in the mitochondrial signalling pathway. Upon induction of apoptosis, Bax undergoes a conformational change and translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol. However, the domains of Bax that are essential for the induction of cytochrome c release are still elusive. Therefore various Bax deletion mutants were generated and expressed in Escherichia coli. The proteins were then purified in order to delineate the function of the transmembrane domain, the BH3 (Bcl-2 homology 3) domain and the putative pore-forming alpha-helices-5 and -6. These proteins were used to analyse the mechanism of Bax-induced cytochrome c release from mitochondria. None of the Bax proteins caused cytochrome c release merely through physical perturbation of the mitochondrial outer membrane. The alpha-helices-5 and -6 of Bax were shown to mediate the insertion of the protein into mitochondrial membranes and to be essential for the cytochrome c -releasing activity of Bax. In contrast, neither the transmembrane domain nor a functional BH3 domain is required for the Bax-mediated release of cytochrome c from mitochondria.

The mechanism of Bax-dependent cytochrome c release is still controversial and may also depend on the actual localisation of cytochrome C: (i) we studied the distribution of cytochrome c in sub-fractions of rat kidney mitochondria and found that 10-20% of the total cytochrome c was associated at the peripheral inner membrane and to some extent organised in the contact sites. (ii) Cytochrome c concentrations in the contact site fractions varied related to surface bound hexokinase activity. It decreased upon reduction of contact sites by glycerol or specific dissociation of the VDAC-ANT complexes by bongkrekate, whereas it increased upon induction of contacts by dextran or association of VDAC-ANT complexes by atractyloside. (iii) The outer membrane pore (VDAC) acquires high capacity for hexokinase binding by interacting with the ANT. Thus, surface-attached hexokinase protein indicated the frequency of VDAC-ANT complexes and the correlation between hexokinase activity and cytochrome c suggested association of the latter to the complexes. (iv) Substances affecting exclusively the structure of either hexokinase (glucose-6P) or cytochrome c (borate) led to a decrease only of the effected protein without changing the concentration of other contact site constituents. (v) Hexokinase was furthermore used as a tool to isolate the contact site forming complex of outer membrane VDAC and inner membrane ANT from Triton-dissolved membranes. Cytochrome c remained attached to the hexokinase VDAC-ANT complexes that were reconstituted in phospholipid vesicles. (vi) The vesicles were loaded with malate and BaxDeltaC released the endogenous cytochrome c from the reconstituted complexes without forming unspecific pores for malate. BaxDeltaC targeted a cytochrome c fraction associated at the VDAC-ANT complex. The cytochrome c organisation was dependent on the actual structure of VDAC and ANT. Thus, the BaxDeltaC effect was suppressed either by hexokinase utilising glucose and ATP or by bongkrekic acid both influencing the pore and ANT structure.

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active creatine kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.

VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator ( ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.

The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-δC released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-δC. (III) The Bax-δC effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-δC effect was as well suppressed by hexokinase phosphorylating glucose.

The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA). It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).

Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intramitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein.

The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C‐terminal truncated oligomeric Bax (BaxΔC). Micromolar concentrations of BaxΔC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxΔC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxΔC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxΔC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase–porin–adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxΔC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxΔC. BaxΔC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. Conclusions: (1) BaxΔC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxΔC action. (3) A limited pool of cytochrome c becomes the first target for BaxΔC.

PURPOSE:Radical-chain mechanisms such as lipid peroxidation are known to show an inverse dose-rate effect, i.e. the radiation effect increases with decreasing dose rate at identical doses applied. The present study was intended to investigate whether an inverse dose-rate effect can be transferred from the level of lipids to the level of proteins.METHOD:Functional inactivation or structural modification by 80kV X-rays of two classes of proteins was investigated: membrane proteins with a natural environment of lipids like the Ca-ATPase of the sarcoplasmic reticulum, succinate dehydrogenase and F0F1-ATPase from the inner mitochondrial membrane. The second class comprises the water-soluble proteins cytosolic creatine kinase (MM-CK) and bovine serum albumin (BSA). Their modification by free radicals of water radiolysis was investigated in the absence and presence of lipid vesicles.RESULTS:For all proteins investigated, an inverse dose-rate effect was observed in the presence of lipids. This also holds for the water-soluble proteins MM-CK and BSA. In the latter two cases, the dose-rate effect disappeared either in the absence of (unsaturated) lipids or in the presence of alpha-tocopherol.CONCLUSION:The largely identical results obtained for a variety of different proteins indicate that inverse dose-rate effects are a normal consequence of radiation induced protein damage in the presence of lipids. In view of the high amount of cellular lipids, this should also hold for the situation in vivo, although due to the comparatively high concentration of intracellular antioxidants the dose-rate dependence might be strongly reduced or even virtually abolished.

Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D(37)-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mi(b)-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mi(b)-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.

Mitochondrial creatine kinase (Mi-CK) occurs in dimeric and octameric forms, bothin vitroandin vivo. The Mi-CK octamer, however, is the predominant formin vivoand is important for various functions of the protein. In the present study we show for the first time a significant decrease of the octamer/dimer ratioin vivo, related to ischemia-induced damage, and a similar decrease of octamer stabilityin vitro, induced by peroxynitrite (PN) radicals. We used animal models to induce ischemia in two different ways: acute ischemia in intact heart (Langendorff perfusion) and chronic ischemiain vivo(LAD-infarction). In both models, impairment of heart function and mitochondrial energy metabolism was associated with a significant decrease of Mi-CK octamer/dimer ratios and of Mi-CK activities. These findings, together with recent data showing that the formation of PN is induced in ischemia and that Mi-CK is a prime target of peroxynitrite (PN)-induced damage, suggest that oxygen radicals generated during ischemia and reoxygenation could be an important factor for the decreased octamer stability. To test this hypothesis, we studied the effect of PN on pure Mi-CKin vitro, both on dissociation of octamers and reassociation of dimers. At 1 mmPN 66% of Mi-CK octamers dissociated into dimers, whereas octamerization of PN-modified dimers was already completely inhibited at 100μmPN. Our data indicate that PN-induced damage could be responsible for the octamer-dimer transition of Mi-CK in ischemia. A loss of Mi-CK octamers would impair the channeling of high energy phosphate out of mitochondria and hence heart function in general.