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 F O F 1 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.

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The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.Keywords: mitochondria; cell death; apoptosis; necrosis; permeability transition; calcium; caspases; fluorescence; probes; cytochrome c.Mitochondria are central to the life of eukaryotic cells. In recent years, it has become clear that mitochondria also play a key role in the pathways to cell death [1±8]. This role of mitochondria cannot be explained by a mere`loss of function' resulting in an energetic deficit. Rather, it is increasingly recognized as aǹ active' process mediated by regulated effector mechanisms in a wide variety of conditions. A large body of observations from the fields of immunology, toxicology, oncology, neurology and cardiology testifies about the centrality of this issue. However, the general reader is probably puzzled by the growing number of conflicting reports, and by the diverging conclusions reached by different groups when mechanistic issues are addressed and/ or cause-relationship effects are discussed. A clear example is represented by the question of cytochrome c release, and of how this event relates to mitochondrial respiration, maintenance of the mitochondrial membrane potential difference (Dc m ) and volume homeostasis.In our opinion, part of the problem resides in the intrinsic difficulties one faces when measuring`mitochondrial function' in intact cells, an issue that is not always adequately appreciated. Mitochondrial responses in vivo may be extremely hard to decipher, and a large discrepancy currently exists between our understanding of mitochondrial function in the isolated organelle and in the intact cell. However, an acceptable description of in vivo events must always take into account facts and concepts established in decades of in vitro studies. Also, it should be realized that discrepancies may arise from inapparent differences in the experimental conditions, but also from artifacts or misinterpretation of the in situ experiments. As should become clear later in the revie...

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to ≈ 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.