The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology.

The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca(2+)-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology.

Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel.

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

The unique histidine in OSCP subunit of F-ATP synthase mediates inhibition of the permeability transition pore by acidic pH.

The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.

Ca2+ binding to F-ATP synthase ß subunit triggers the mitochondrial permeability transition.

F-ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca2+-dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the "Ca2+-trigger site" of the PTP to the catalytic site of the F-ATP synthase ß subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the ß subunit, which show a selective decrease in Ca2+-ATP hydrolysis, confer resistance to Ca2+-induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death.

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits.

BACKGROUND/AIMS: The permeability transition pore (PTP) is an unselective, Ca2+-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca2+-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. METHODS: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. RESULTS: Null mutants for the subunits e and g display dimer formation upon Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. CONCLUSION: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.

From ATP to PTP and Back: A Dual Function for the Mitochondrial ATP Synthase.

Mitochondria not only play a fundamental role in heart physiology but are also key effectors of dysfunction and death. This dual role assumes a new meaning after recent advances on the nature and regulation of the permeability transition pore, an inner membrane channel whose opening requires matrix Ca(2+) and is modulated by many effectors including reactive oxygen species, matrix cyclophilin D, Pi (inorganic phosphate), and matrix pH. The recent demonstration that the F-ATP synthase can reversibly undergo a Ca(2+)-dependent transition to form a channel that mediates the permeability transition opens new perspectives to the field. These findings demand a reassessment of the modifications of F-ATP synthase that take place in the heart under pathological conditions and of their potential role in determining the transition of F-ATP synthase from and energy-conserving into an energy-dissipating device.

F-ATPase of Drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca2+-induced Ca2+ release.

Mitochondria of Drosophila melanogaster undergo Ca(2+)-induced Ca(2+) 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 Ca(2+) 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 Mg(2+)/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R(+) cells sensitizes the mCrC to Ca(2+) 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 Ca(2+) and thiol oxidants and inhibited by Mg(2+)/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.

Dimers of mitochondrial ATP synthase form the permeability transition pore.

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

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 Ca(2+)-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca(2+) ionophore ETH129, which allows electrophoretic Ca(2+) 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.

Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage.

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.

The oligomycin-sensitivity conferring protein of mitochondrial ATP synthase: emerging new roles in mitochondrial pathophysiology.

The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F(O)F1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between F(O) and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²âº required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²âº-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.

Ectopic F0F 1 ATP synthase contains both nuclear and mitochondrially-encoded subunits.

Over the past few years, several reports have described the presence of F0F1 ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F1 sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F0F1 complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F0F1 complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.

Cyclophilin D in Mitochondrial Dysfunction: A Key Player in Neurodegeneration?

Mitochondrial dysfunction plays a pivotal role in numerous complex diseases. Understanding the molecular mechanisms by which the "powerhouse of the cell" turns into the "factory of death" is an exciting yet challenging task that can unveil new therapeutic targets. The mitochondrial matrix protein CyPD is a peptidylprolyl cis-trans isomerase involved in the regulation of the permeability transition pore (mPTP). The mPTP is a multi-conductance channel in the inner mitochondrial membrane whose dysregulated opening can ultimately lead to cell death and whose involvement in pathology has been extensively documented over the past few decades. Moreover, several mPTP-independent CyPD interactions have been identified, indicating that CyPD could be involved in the fine regulation of several biochemical pathways. To further enrich the picture, CyPD undergoes several post-translational modifications that regulate both its activity and interaction with its clients. Here, we will dissect what is currently known about CyPD and critically review the most recent literature about its involvement in neurodegenerative disorders, focusing on Alzheimer's Disease and Parkinson's Disease, supporting the notion that CyPD could serve as a promising therapeutic target for the treatment of such conditions. Notably, significant efforts have been made to develop CyPD-specific inhibitors, which hold promise for the treatment of such complex disorders.

The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species.

The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca2+-activated, high-conductance channels in the inner membrane of mitochondria from a variety of eukaryotes led to renewed interest in the permeability transition (PT), a permeability increase mediated by the PT pore (PTP). The PT is a Ca2+-dependent permeability increase in the inner mitochondrial membrane whose function and underlying molecular mechanisms have challenged scientists for the last 70 years. Although most of our knowledge about the PTP comes from studies in mammals, recent data obtained in other species highlighted substantial differences that could be perhaps attributed to specific features of F-ATP synthase and/or ANT. Strikingly, the anoxia and salt-tolerant brine shrimp Artemia franciscana does not undergo a PT in spite of its ability to take up and store Ca2+ in mitochondria, and the anoxia-resistant Drosophila melanogaster displays a low-conductance, selective Ca2+-induced Ca2+ release channel rather than a PTP. In mammals, the PT provides a mechanism for the release of cytochrome c and other proapoptotic proteins and mediates various forms of cell death. In this review, we cover the features of the PT (or lack thereof) in mammals, yeast, Drosophila melanogaster, Artemia franciscana and Caenorhabditis elegans, and we discuss the presence of the intrinsic pathway of apoptosis and of other forms of cell death. We hope that this exercise may help elucidate the function(s) of the PT and its possible role in evolution and inspire further tests to define its molecular nature.

The mitochondrial permeability transition: Recent progress and open questions.

Major progress has been made in defining the basis of the mitochondrial permeability transition, a Ca2+ -dependent permeability increase of the inner membrane that has puzzled mitochondrial research for almost 70 years. Initially considered an artefact of limited biological interest by most, over the years the permeability transition has raised to the status of regulator of mitochondrial ion homeostasis and of druggable effector mechanism of cell death. The permeability transition is mediated by opening of channel(s) modulated by matrix cyclophilin D, the permeability transition pore(s) (PTP). The field has received new impulse (a) from the hypothesis that the PTP may originate from a Ca2+ -dependent conformational change of F-ATP synthase and (b) from the reevaluation of the long-standing hypothesis that it originates from the adenine nucleotide translocator (ANT). Here, we provide a synthetic account of the structure of ANT and F-ATP synthase to discuss potential and controversial mechanisms through which they may form high-conductance channels; and review some intriguing findings from the wealth of early studies of PTP modulation that still await an explanation. We hope that this review will stimulate new experiments addressing the many outstanding problems, and thus contribute to the eventual solution of the puzzle of the permeability transition.

The mitochondrial chaperone TRAP1 regulates F-ATP synthase channel formation.

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

Cyclophilin D in mitochondrial pathophysiology.

Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.

The Influence of the Type of Dry-Cured Italian PDO Ham on Cathepsin B Activity Trend during Processing.

Cathepsin B activity was measured during processing in hams originating from the main Italian prosciutto PDOs: Parma, San Daniele and Toscano. Sixty-five heavy pig thighs, from sixty-five Italian large white x Italian Landrace pigs bred and slaughtered in the same conditions were considered. Five thighs represented the post-mortem control time. The other 60 were distributed one plant per PDO, following a balanced plan. The thighs were sampled at the biceps femoris in groups of four per plant in the following ripening phases: salting, resting, drying, greasing, end of curing. The activity of the Cathepsin B (U/g protein) was determined by means of fluorescence measurements. The Cathepsin B ripening trend of the various PDOs was significantly different, particularly during the initial and mid-curing stage. This activity correlates with the proteolysis index through a PDO dependent pattern, indicating that different processing conditions can influence the quality of prosciutto, since they determine its biochemical development.

The f subunit of human ATP synthase is essential for normal mitochondrial morphology and permeability transition.

The f subunit is localized at the base of the ATP synthase peripheral stalk. Its function in the human enzyme is poorly characterized. Because full disruption of its ATP5J2 gene with the CRISPR-Cas9 strategy in the HAP1 human model has been shown to cause alterations in the amounts of other ATP synthase subunits, here we investigated the role of the f subunit in HeLa cells by regulating its levels through RNA interference. We confirm the role of the f subunit in ATP synthase dimer stability and observe that its downregulation per se does not alter the amounts of the other enzyme subunits or ATP synthase synthetic/hydrolytic activity. We show that downregulation of the f subunit causes abnormal crista organization and decreases permeability transition pore (PTP) size, whereas its re-expression in f subunit knockdown cells rescues mitochondrial morphology and PTP-dependent swelling.

Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.