Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.     

F-ATPase of Drosophila melanogaster Forms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+ Release

Mitochondria of Drosophila melanogaster undergo Ca²⁺-induced Ca²⁺ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca²⁺ and H⁺. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg²⁺/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S₂R⁺ cells sensitizes the mCrC to Ca²⁺ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca²⁺ and thiol oxidants and inhibited by Mg²⁺/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

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

The permeability transition pore (PTP) is a Ca²⁺-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 Ca²⁺ 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 Mg²⁺. By studying the effects of Mg²⁺, cyclosporin A (CsA) and ubiquinone 0 (Ub₀) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg²⁺ and Ub₀ 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.     

Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore

Triggering ofthe permeability transition pore (PTP) in isolated mitochondria causesrelease of matrix Ca²⁺, ions, andmetabolites, and it has been proposed that the PTP mediatesmitochondrial Ca²⁺ release inintact cells. To study the role of the PTP in mitochondrial energymetabolism, the mitochondrial content ofCa²⁺,Mg²⁺, ATP, and ADP was determinedin hormonally stimulated rat livers perfused with cyclosporin A (CsA).Stimulation of livers perfused in the absence of CsA with glucagon andphenylephrine induced an extensive uptake ofCa²⁺,Mg²⁺, and ATP plus ADP by themitochondria, followed by a release on omission of hormones. In thepresence of CsA, the PTP was fully inhibited, but neither thehormone-induced uptake of Ca²⁺,ATP, or ADP by mitochondria nor their release after washout of hormoneswas significantly changed. We conclude that the regulation of sustainedchanges in mitochondrial Ca²⁺content induced by hormonal stimulation is independent of the PTP.

     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.     

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca<Superscript>2+</Superscript>-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca²⁺-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys⁵⁶ relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca²⁺-induced ANT conformational change and mitochondrial swelling. ANT-Pro⁶¹ isomerization increased ANT-Cys⁵⁶ relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca²⁺ induced ANT “c” conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca²⁺-induced ANT “c” conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca²⁺ induces the ANT “c” conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.     

Study on the relationship between calcium-induced calcium release from mitochondria and PTP opening

The permeability transition pore (PTP) is central for mitochondria function. PTP either open in low-conductance state to carry out mCICR (Ca²⁺-induced Ca²⁺ release from mitochondria) and play roles in cell phsyiological activities or open in high-conductance conformation to release harmful substances and play important roles in cell pathological responses and apoptosis. The results of study on the relationship between mCICR and PTP opening show Ca²⁺ concentrations but not the Ca²⁺ delivery mode determined the occurrence of mCICR or PTP opening. Ca²⁺-induced PTP opening began with and depended on mCICR. mCICR was a prerequisite for H₂O₂ and As₂O₃-induced PTP opening. The results indicated that the PTP opening was determined by Ca²⁺ stimulation intensity but not mode. PTP could switch from low- to high-conductance conformation and the PTP open by high-conductance began with low-conductance state. mCICR is necessary for Ca²⁺-dependent PTP opening. Our data suggested also that it would be possible to control cellular responses first by modulating mCICR, then by regulating PTP opening.     

Reactive oxygen species and permeability transition pore in rat liver and kidney mitoplasts

Mitochondrial permeability transition is typically characterized by Ca²⁺ and oxidative stress-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A, known as the permeability transition pore (PTP). Data from our laboratory provide evidence that the PTP is formed when inner membrane proteins aggregate as a result of disulfide cross-linking caused by thiol oxidation. Here we compared the redox properties between PTP in intact mitochondria and mitoplasts. The rat liver mitoplasts retained less than 5% and 10% of the original outer membrane markers monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase. In line with the redox nature of the PTP, mitoplasts that were more susceptible to PTP opening than intact mitochondria showed higher rates of H₂O₂ generation and decreased matrix NADPH-dependent antioxidant activity. Mitoplast PTP was also sensitive to the permeability transition inducer tert-butyl hydroperoxide and to the inhibitors cyclosporin A, EGTA, ADP, dithiothreitol and catalase. Taken together, these data indicate that, in mitoplasts, PTP exhibits redox regulatory characteristics similar to those described for intact mitochondria.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

Protection of pancreatic INS-1 ��-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin

Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca²⁺-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca²⁺ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca²⁺ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca²⁺ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Characteristics and possible functions of mitochondrial Ca transport mechanisms

Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca²⁺]_m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca²⁺]_m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca²⁺ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca²⁺ pulses or transients. Mitochondria can also sequester Ca²⁺ from these transients so as to modify the shape of Ca²⁺ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca²⁺ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca²⁺ transport and a discussion of how they might function are described in this paper.     

Cyclosporin A binding in mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury

When loaded with high (pathological) levels of Ca²⁺, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca²⁺], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca²⁺] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca²⁺-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)     

mCICR is required for As2O3-induced permeability transition pore opening and cytochrome c release from mitochondria

The permeability transition pore (PTP) is central for apoptosis by acting as a good candidate pathway for the release of Cyt. c and apoptosis induction factors (AIF). Arsenite induces apoptosis via a direct effect on PTP. To characterize the exact mechanism for arsenite induces PTP opening, the effect of Ca²⁺ on As₂O₃-induced PTP opening, the relationship between As₂O₃-induced PTP opening and Cyt. c release from mitochondria and calcium-induced calcium release from mitochondria (mCICR), and the effects of As₂O₃ on Ca²⁺-induced PTP opening were studied. The results showed As₂O₃ induces Cyt. c release by triggering PTP opening. Ca²⁺ is necessary for As₂O₃-induced PTP opening. As₂O₃-induced PTP opening and Cyt. c release depends on mCICR. As₂O₃ promotes PTP opening by lowering Ca²⁺-threshold. These results indicated As₂O₃ induce Cyt. c release from mitochondria by lowering Ca²⁺-threshold for PTP and triggering mCICR-dependent PTP opening. Suggesting that it is possible to control apoptosis by altering Ca²⁺ threshold and mCICR to modulate PTP opening and Cyt. c release.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.     

Mitochondrial ATP synthase inhibitory factor 1 interacts with the p53–cyclophilin D complex and promotes opening of the permeability transition pore

The mitochondrial permeability transition pore (PTP) is a Ca²⁺-dependent megachannel that plays an important role in mitochondrial physiology and cell fate. Cyclophilin D (CyPD) is a well-characterized PTP regulator, and its binding to the PTP favors pore opening. It has previously been shown that p53 physically interacts with CyPD and opens the PTP during necrosis. Accumulating studies also suggest that the F-ATP synthase contributes to the regulation and formation of the PTP. F-ATP synthase IF1 (mitochondrial ATP synthase inhibitory factor 1) is a natural inhibitor of F-ATP synthase activity; however, whether IF1 participates in the modulation of PTP opening is basically unknown. Here, we demonstrate using calcium retention capacity assay that IF1 overexpression promotes mitochondrial permeability transition via opening of the PTP. Intriguingly, we show that IF1 can interact with the p53–CyPD complex and facilitate cell death. We also demonstrate that the presence of IF1 is necessary for the formation of p53–CyPD complex. Therefore, we suggest that IF1 regulates the PTP via interaction with the p53–CyPD complex, and that IF1 is necessary for the inducing effect of p53–CyPD complex on PTP opening.     

Non-conventional mitochondrial permeability transition: Its regulation by mitochondrial dynamics

Mitochondrial permeability transition (MPT) is a phenomenon that the inner mitochondrial membrane (IMM) loses its selective permeability, leading to mitochondrial dysfunction and cell injury. Electrophysiological evidence indicates the presence of a mega-channel commonly called permeability transition pore (PTP) whose opening is responsible for MPT. However, the molecular identity of the PTP is still under intensive investigations and debates, although cyclophilin D that is inhibited by cyclosporine A (CsA) is the established regulatory component of the PTP. PTP can also open transiently and functions as a rapid mitochondrial Ca²⁺ releasing mechanism. Mitochondrial fission and fusion, the main components of mitochondrial dynamics, control the number and size of mitochondria, and have been shown to play a role in regulating MPT directly or indirectly. Studies by us and others have indicated the potential existence of a form of transient MPT that is insensitive to CsA. This “non-conventional” MPT is regulated by mitochondrial dynamics and may serve a protective role possibly by decreasing the susceptibility for a frequent or sustained PTP opening; hence, it may have a therapeutic value in many disease conditions involving MPT.     

Ursodeoxycholic acid,in contrast to other bile acids,induces Ca<Superscript>2+</Superscript>-dependent cyclosporin A-insensitive permeability transition in liver mitochondria in the presence of potassium chloride

The effects of hydrophobic and hydrophilic bile acids as inducers of Ca²⁺-dependent permeability of the inner membrane were studied on isolated liver mitochondria. It is shown that in the absence of the inorganic phosphate (P_i)–a modulator of the mitochondrial pore–hydrophobic bile acids (lithocholic, deoxycholic, chenodeoxycholic) at concentrations of 20–50 μM, as well as a hydrophilic cholic acid at a concentration of 800 μM, induce swelling of liver mitochondria loaded with Ca²⁺. This effect is completely eliminated by a specific inhibitor of mitochondrial pore cyclosporin A (CsA). The effect of the bile acids as inducers of Ca²⁺-dependent CsA-sensitive mitochondrial pore is not associated with the modulation of the P_i effects. In contrast to other tested bile acids, a hydrophilic ursodeoxycholic acid (UDCA) at a concentration of 400 μM is able to induce Ca²⁺-dependent CsA-sensitive pore opening in liver mitochondria only in the presence of P_i or in the absence of potassium chloride in the incubation medium. In the presence of potassium chloride but in the absence of P_i, UDCA effects associated with the induction of the inner membrane permeability (swelling of mitochondria, drop in Δψ, and Ca²⁺ release from the matrix) are also observed in the presence of CsA. This Ca²⁺-dependent permeability of the inner membrane, in contrast to the “classical” CsA-sensitive pore, is characterized by a lower intensity of the mitochondrial swelling, a total drop in Δψ, and Ca²⁺ release from the matrix and is blocked by P_i. We suggest that the induction of the CsA-insensitive permeability in the inner mitochondrial membrane by UDCA is associated with activation of electrophoretic influx of K⁺ into the matrix and Ca²⁺ release from the matrix in exchange to H+. The effect of P_i as a blocker of such permeability is discussed.