Mitochondrial Membrane Pore Research Articles

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118-125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.

Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins.

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.

N-methyl-d-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-d-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca(2+) antagonist; 3) LaCl(3), an extracellular Ca(2+) channel blocker; 4) diphenyleiodonium, a heme-ferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca(2+) dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria.

In Situ Imaging of Mitochondrial Outer-Membrane Pores using Atomic Force Microscopy

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.

Calcium-Dependent Nonspecific Permeability of the Inner Mitochondrial Membrane Is Not Induced in Mitochondria of the Yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

The NAD+ precursor nicotinamide governs neuronal survival during oxidative stress through protein kinase B coupled to FOXO3a and mitochondrial membrane potential.

Nicotinamide, a beta-nicotinamide adenine dinucleotide (NAD) precursor and an essential nutrient for cell growth and function, may offer critical insights into the specific cellular mechanisms that determine neuronal survival, since this agent significantly impacts upon both neuronal and vascular integrity in the central nervous system. The authors show that nicotinamide provides broad, but concentration-specific, protection against apoptotic genomic DNA fragmentation and membrane phosphatidylserine exposure during oxidative stress to secure cellular integrity and prevent phagocytic cellular demise. Activation of the protein kinase B (Akt1) pathway is a necessary requirement for nicotinamide protection, because transfection of primary hippocampal neurons with a plasmid encoding a kinase-deficient dominant-negative Akt1 as well as pharmacologic inhibition of phosphatidylinositol-3-kinase phosphorylation of Akt1 eliminates cytoprotection by nicotinamide. Nicotinamide fosters neuronal survival through a series of intimately associated pathways. At one level, nicotinamide directly modulates mitochondrial membrane potential and pore formation to prevent cytochrome c release and caspase-3-and 9-like activities through mechanisms that are independent of the apoptotic protease activating factor-1. At a second level, nicotinamide maintains an inhibitory phosphorylation of the forkhead transcription factor FOXO3a at the regulatory sites of Thr and Ser and governs a unique regulatory loop that prevents the degradation of phosphorylated FOXO3a by caspase-3. Their work elucidates some of the unique neuro-protective pathways used by the essential cellular nutrient nicotinamide that may direct future therapeutic approaches for neurodegenerative disorders.

The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis.

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.

The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis.

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.

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.

Overcoming drug resistance: targeting more than one site

Activation of the molecular machinery of programmed cell death (apoptosis) is a convergence point for many cytotoxic agents, irrespective of the primary mechanism of action. Indeed, apoptosis is induced in response to DNA strand breaks caused by such mechanistically diverse anti-leukemic drugs as the nucleoside cytosine arabinoside (ara-C), anthracyclines (daunorubicin, idarubicin), and agents directed against topoisomerase I (camptothecins) and II (etoposide, mitoxantrone). The identification of apoptosis on both the molecular and cellular levels has led to the finding that the mitochondrion plays a pivotal role in governing the decision and execution of the apoptotic process (reviewed in [1]). It is now clear that the mitochondrion acts as a repository for diverse proand anti-apoptotic proteins, many of which are located in mitochondrial membranes at the interface between the organelle and the cytosol [2,3]. In the response to cellular stress, such as from genotoxic anti-cancer agents, changing the mitochondrial membrane potential and pore size releases mitochondrial factors including cytochrome c and apoptosis inducing factor (AIF). These factors, in turn, lead to the enhanced caspase activity that results in cleavage of critical cellular substrates [1–3]. This pro-apoptotic pathway is kept in check by two protein groups— namely, the BCL-2 protein family in the mitochondrion and inhibitor of apoptosis (IAP) family in the cytosol. The balance between these proand anti-apoptotic components influences the ultimate cellular decision between life and death. The development of new anti-cancer agents is based on the premise that therapeutic ratios can be favorably altered by understanding and exploiting differences between normal and malignant cells. In this regard, mitochondrial membrane components seem like ripe targets to exploit for the purpose of enhancing the cell’s ‘stress response’ to chemotherapy. Of particular interest, are components that determine membrane pore size and permeability—the so-called mitochondrial permeability pore transition complex (PTPC) that determines whether or not cytochrome c will be released in response to a potentially cytotoxic stimulus [5]. One such target for manipulation is the peripheral benzodiazepine receptor (pBzR) [6], an outer mitochondrial membrane protein that is a PTPC constituent and acts similarly to BCL-2 and BCL-XL by interacting with the pore-forming protein voltage-dependent anion channel (VDAC) in a way that keeps the channel closed, thereby blocking mitochondrial permeabilization and abrogating apoptosis [3–6]. Importantly, pBzRs are expressed in both normal and leukemia hematopoietic cells and can protect those cells from undergoing apoptosis in response to oxidative stress [7]. While the apoptotic machinery is the final determinant of stress-induced programmed cell death, malignant cells call into action multiple factors from the cell surface to the genome that confer an uncanny ability to survive in an adverse and stressful environment. One such factor is the multidrug resistance protein known as MDR-1 or P-glycoprotein (PgP), a membrane transport pump located at the cell surface that promotes efflux of exogenous toxins and prevents intracellular accumulation at high-enough concentrations or for long-enough periods of time to inflict lethal damage (reviewed in [8]). Tumor cell MDR-1 expression has been linked to clinical drug resistance and poor clinical outcome in diverse malignancies, including acute myelogenous leukemias (AMLs). The fact that anti-AML anthracyclines and etoposide are MDR-1 substrates and the availability of agents that inhibit MDR-1 activity have led to numerous trials of MDR-1 inhibitors in AML, particularly in * Corresponding author.

Tumor Necrosis Factor-α Induces Bax-Bak Interaction and Apoptosis, Which Is Inhibited by Adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.

The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L).

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.

Ibuprofen-induced liver mitochondrial permeability transition

This study examined the effect of the nonsteroidal anti-inflammatory drug ibuprofen on liver inner mitochondrial membrane permeability transition in the presence of Ca 2+ and phosphate. Incubation of isolated liver mitochondria with ibuprofen (0.1–0.4 mM) induced inner mitochondrial membrane permeability as indicated by loss of inner mitochondrial membrane potential, swelling of matrix and loss of pre-accumulated Ca 2+. The presence of cyclosporin A (1 μM) in the incubation medium preventing ibuprofen from causing loss of inner mitochondrial membrane potential, swelling and loss of pre-accumulated Ca 2+. It is concluded that ibuprofen acted as an activator of Ca 2+ and phosphate in promoting the opening of inner mitochondrial membrane pore.

Mitochondrial permeability in neuronal death: possible relevance to the pathogenesis of Parkinson's disease

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes catecholaminergic nerve cell loss and a syndrome similar to Parkinson's disease (PD). The metabolite of MPTP, MPP(+) (1-methyl-4-phenylpyridinium), decreases mitochondrial complex I activity similar to that in the PD nigra. Opening of a multi-protein, mitochondrial membrane pore constitutes a critical decisional event in some forms of apoptosis. We review recent findings showing that the permeability transition pore (PTP) opening caused by a decrease in the mitochondrial membrane potential (DeltaPsi(M)) contributes to MPP(+)-induced apoptosis. The reduction in DeltaPsi(M) appears to result from decreased proton pumping at complex I and therefore decreased complex I activity may also contribute to apoptosis in PD.

Salicylate-induced kidney mitochondrial permeability transition is prevented by cyclosporin A

The effect of salicylate, the active metabolite of aspirin (acetyl salicylic acid) in the presence of Ca 2+ and phosphate on mitochondrial permeability transition (MPT) was studied. MPT is often associated with opening of a Ca 2+-induced pore. The opening of this pore leads to swelling, loss of mitochondrial membrane potential and release of accumulated Ca 2+. In freshly isolated rat kidney mitochondria, salicylate (400 μM) in the presence of 20 nmol Ca 2+/mg protein and 0.1 mM phosphate induced swelling, loss of mitochondrial membrane potential and release of accumulated Ca 2+. All these changes were eliminated when cyclosporin A (1 μM), (a pore inhibitory agent) was included in the incubation medium. Unlike salicylate, unhydrolyzed aspirin (400 μM) induced these changes slightly. We concluded that salicylate acts as an activator of Ca 2+ and phosphate in promoting the opening of kidney inner mitochondrial membrane pore. As a result a great consideration should be given to its toxicological effect.

Calcium signaling and cytotoxicity.

The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery.

Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The M r suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCl. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca 2+ between 100 and 600 μM. The liberation of malate or ATP by Ca 2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.

On the interactions of Ca2+ and cyclosporin A with a mitochondrial inner membrane pore: a study using cobaltammine complex inhibitors of the Ca2+ uniporter.

The mitochondrial inner membrane contains a Ca(2+)-activated pore of possible relevance to the pathogenesis of ischaemia/reperfusion injury which is inhibited by the immunosuppressant cyclosporin A (CSA). The present study employs a number of novel cobaltammine complex inhibitors of the Ca2+ uniporter (mediating Ca2+ uptake) to examine whether intramitochondrial Ca2+ influences the capacity of CSA to block the pore. Using dissipation of the inner membrane potential as a means of monitoring the state of the pore, it is shown that CSA blockade is facilitated as Ca2+ uptake is restricted. Ca2+ also depresses and reverses the binding of [3H]CSA to mitochondria, but Ca2+ is ineffective when its uptake is prevented. It is concluded that a high intramitochondrial Ca2+ concentration antagonizes pore inhibition by CSA. The significance of this is discussed.

Effect of macromolecules on the regulation of the mitochondrial outer membrane pore and the activity of adenylate kinase in the inter-membrane space

Macromolecules as components of the physiological mitochondrial environment were substituted by dextrans of different molecular weight. The addition of 10% dextran (molecular weights varying between 20 and 500 kDa) affected neither basic mitochondrial parameters (state 4 and state 3 respiration) nor kinetic properties of soluble kinases. A significant increase by 10% dextran was however observed of the voltage sensitivity of isolated porin when reconstituted in planar bilayers. The pores adapted the low conducting state already at a voltage of 10 mV. This effect of the macromolecules may explain the higher diffusion resistance of adenine nucleotides across the outer membrane as observed in different experiments: (i) the Michaelis constant of adenylate kinase in the inter-membrane space increased, in contrast to the soluble enzyme, from 118 ± 10 μM to 193 ± 20 μM ADP, (ii) in the presence of competing external pyruvat kinase, the mitochondrial utilization of ADP, produced by adenylate kinase in the inter-membrane space, was improved 3-fold suggesting a reduced ADP diffusion out of the outer mitochondrial compartment. The influence of the various dextrans correlated with the increase in molecular weight of the dextrans. The effect on the kinetic constants was dependent on the dextran concentration in terms of weight and not of molarity. The oncotic pressure and viscosity of dextran solutions with different molecular weight showed a comparable dependence. In general, the data indicate that the outer membrane pore responds to an increased oncotic pressure by reducing adenine nucleotide permeability. This suggests the physiological existence of a third adenine nucleotide compartment between the two envelope membranes which may be important especially at high metabolic fluxes.