Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

Copper induces permeability transition through its interaction with the adenine nucleotide translocase

In this work we examined the effect of low concentrations of Cu(2+) on the opening of the mitochondrial non-specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca(2+) efflux and mitochondrial swelling. The experimental results indicate that Cu(2+) at very low concentrations promoted the release of accumulated Ca(2+), as well as mitochondrial swelling, provided 1,10-phenanthroline has been added. Carboxyatractyloside and Cu(2+) exhibited additive effects on these parameters. After Cu(2+) titration of membrane thiols, it might be assumed that the blockage of 5.9nmol of SH/mg protein suffices to open the non-specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N-ethylmaleimide inhibited the Cu(2+)-induced Ca(2+) efflux, it is proposed that the target site for Cu(2+) is located in the ADP/ATP carrier.     

Ca binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca²⁺-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys⁵⁶ relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca²⁺ and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca²⁺ interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys⁵⁶ residue increasing its relative mobility. The binding of ADP that stabilizes the conformation “m” of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys⁵⁶, accounting for reducing its relative mobility. The results suggest that Ca²⁺ binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys⁵⁶ relative mobility and that this may constitute a potential critical step of Ca²⁺-induced PTP opening.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K _eq ^app) of this reaction. Subsequent excess of over the K _eq ^app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K _eq ^app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K _eq ^app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.     

Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.     

Oligomycin strengthens the effect of cyclosporin A on mitochondrial permeability transition by inducing phosphate uptake

The purpose of this work was to assess the effect of oligomycin on the mitochondrial membrane permeability transition. The antibiotic was found to strengthen cyclosporin A (CSA)-induced protection of non-specific permeability, which is triggered by a matrix Ca2+ load in the absence of ADP. Oligomycin also reinforced the protective effect of CSA on carboxyatractyloside-induced pore opening in the absence of ADP, but failed to do so in mitochondria incubated under anaerobic conditions or after addition of CCCP. Analyzing the efflux of matrix Ca2+, we found that mitochondrial swelling and the collapse of the transmembrane electric gradient coincided with membrane leakage. The effects of the antibiotic were observed in phosphate-containing media but not in the presence of acetate. Furthermore, N-ethylmaleimide hindered the protective effect of oligomycin-CSA. In addition, the matrix phosphate concentration increased concurrently with a diminution in the matrix-free fraction of Ca2+. We concluded that oligomycin increases phosphate uptake by stimulating the phosphate-/OH- exchange reaction.     

The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis

Current research on the mitochondrial permeability transition pore (PTP) and its role in cell death faces a paradox. Initially considered as an in vitro artifact of little pathophysiological relevance, in recent years the PTP has received considerable attention as a potential mechanism for the execution of cell death. The recent successful use of PTP desensitizers in several disease paradigms leaves little doubt about its relevance in pathophysiology; and emerging findings that link the PTP to key cellular signalling pathways are increasing the interest on the pore as a pharmacological target. Yet, recent genetic data have challenged popular views on the molecular nature of the PTP, and called into question many early conclusions about its structure. Here we review basic concepts about PTP structure, function and regulation within the framework of intracellular death signalling, and its role in disease pathogenesis.     

Possible role of amyloid-beta,adenine nucleotide translocase and cyclophilin-D interaction in mitochondrial dysfunction of Alzheimer's disease

Alzheimer''s disease (AD) is a common neurodegenerative disease characterized by both extra- as well as intracellular deposition of amyloid beta peptides (Aβ). The accumulation of Aβ in mitochondria is associated with mitochondrial dysfunction and oxidative stress in AD. Recent evidences suggest the involvement of Aβ interaction with mitochondrial proteins such as cyclophilin-D (CypD) in oxidative stress, mitochondrial permeability transition (MPT) and Alzheimer''s associated neurodegeneration. The present study is an effort to elucidate the molecular interaction of Aβ with other proteins involved in MPT like adenine nucleotide translocase (ANT). Based on our prediction for sub-cellular localization using WolfPSORT and other experimental evidences, we suggest that Aβ molecules localize in mitochondrial inner membrane in close vicinity with ANT. Our simulation study for protein–protein interaction clearly suggests that the ANT-Aβ interaction is stronger than CypD-Aβ interaction. Further the lipophilic nature and evidences regarding the localization of Aβ in the mitochondrial inner-membrane also support the possibility of strong interaction between ANT and Aβ. Interaction between ANT and Aβ may affect normal physiological function of ANT i.e. transport of ATP and ADP. Since both the CypD-Aβ as well as ANT-Aβ interaction are energetically favorable and both CypD and ANT are associated with the regulation of MPT, the functional impact of both these interactions warrants more in-depth investigations for elucidating the mechanisms involved in Aβ-induced oxidative stress.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.     

Proton permeation, adenine nucleotide translocation and respiratory control in mitochondria cross-linked by dimethylsuberimidate

Rat liver mitochondria or isolated mitoplasts were treated with the cross-linking agent, dimethylsuberimidate, under conditions (pH 7.5; 0°C) which were not detrimental for the coupling quality of the mitochondria and the effect was evaluated on a kinetic basis. When about 25% of the NH₂-groups reacted, the mitochondria or the mitoplasts acquired complete osmotic stability. Succinate oxidation in state 4 was inhibited by about 30–35%. This effect was also observed when the organelles were amidinated by methylacetimidate, a monofunctional imidate which caused no osmotic stabilization. Uncouplers stimulated succinate oxidation in cross-linked mitochondria to the same extent as in the control, in contrast stimulation by ADP was suppressed. Accordingly, the rate of decay of the respiration-dependent cross-membrane proton gradient was only decreased by 25%, whereas the ATPase and adenine nucleotide translocase were strongly inhibited. In the cross-linked mitochondria, the extent of inhibition of the ATPase and of the translocase was found to be the same whether the assays were performed at 30°C (like the respiratory assay) or at 0°C. The effect of methylacetimidate treatment on these activities at the two temperatures was different. At 30°C, the ATPase was not inhibited and the extent of inhibition of ATP translocation was small. At 0°C, the two activities were nearly as much inhibited as in cross-linked mitochondria. Our results suggest that a considerable rigidity can be introduced in the coupling membrane by cross-linking, without a major loss in the initial step of energy conservation. However, the energy conserved in the proton gradient cannot be utilized for ATP synthesis, probably because of the restricted mobility of adenine nucleotide translocase in the cross-linked mitochondria.     

Cryopreservation induces mitochondrial permeability transition in a bovine sperm model

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 10⁶ mL⁻¹ and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca²⁺ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.     

Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide

It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity. Our results show that: (i) norbormide isomers affect PTP in a rat-selective fashion; however, no relevant differences between lethal and non-lethal forms are observed suggesting that drug regulation of PTP-activity and lethality in rats are unrelated phenomena; (ii) a (phenylvinyl)pyridine moiety represents the key element conferring the PTP-activating effect; (iii) cationic derivatives of rat-active compounds accumulate in the matrix via the membrane potential and activate the PTP also in mouse and guinea pig mitochondria. These findings suggest that the norbormide-sensitive PTP-target is present in all species examined, and is presumably located on the matrix side. The species-selectivity may depend on the unique properties of a transport system allowing drug internalisation in rat mitochondria.     

A novel adenine nucleotide translocase inhibitor,MT-21, induces cytochrome c release by a mitochondrial permeability transition-independent mechanism

The release of cytochrome c from mitochondria is a critical step during apoptosis. In order to study this process, we have used a synthetic compound, MT-21, that is able to initiate release of cytochrome c from isolated mitochondria. We demonstrate that MT-21 significantly inhibits ADP transport activity in mitochondria and reduces binding of the adenine nucleotide translocase (ANT) to a phenylarsine oxide affinity matrix. These results suggest that ANT, one of the components of the mitochondrial permeability transition (PT) pore, is the molecular target for MT-21. In agreement with this, the MT-21-induced cytochrome c release was effectively inhibited in the presence of ANT ligands, and MT-21 could dissociate ANT from a complex with a glutathione S-transferase-cyclophilin D fusion protein. Interestingly, we also found that specific inhibitors of ANT such as MT-21 and atractyloside could induce cytochrome c release without mitochondrial swelling and that this event was highly dependent on the presence of Mg(2+). These results suggest that although ANT resides in the mitochondrial inner membrane, specific ANT inhibitors can induce cytochrome c release without having an effect on inner membrane permeability. Therefore, MT-21 can be a powerful tool for studying the mechanism of PT-independent cytochrome c release from mitochondria.