The permeability transition pore as a Ca(2+) release channel: new answers to an old question

Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals.     

Ca(2+)-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress

Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca(2+), energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca(2+). Ca(2+) uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca(2+) (but not Mg(2+)) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)-an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca(2+) ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca(2+) concentration and required Ca(2+) localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca(2+) transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.     

Relese of Ca2+ from mitochondria after mitochondrial membrane depolarisation

With the aid of specific inhibitors of Ca(2+)-uniporter (ruthenium red) and mitochondrial permeability transition pore, PTP (cyclosporine A) it is shown that PTP opening takes place after loading the rat liver mitochondria with calcium and depolarisation of mitochondrial membrane with protonophore (carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and the pore opening accounts for accelerated efflux of calcium from mitochondrial matrix as well as availability of "rapid" component of two-exponential kinetic curve of Ca(2+)-efflux. An analysis of kinetic data of Ca2+ transport after membrane depolarisation also confirms our earlier observations that time frame of the pore open state is restricted, and membrane integrity is restored before all the calcium load is delivered into incubation medium. The absence of additivity between the shares of Ca(2+)-uniporter and PTP in Ca(2+)-transport is observed, and conclusion is made that partial share of PTP in calcium transport is not a constant, but a variable constituent which is diminished to zero as soon as the Ca(2+)-uniporter activity reaches its maximum after the abolition of membrane potential with CCCP. Based on some observations, it is supposed also that PTP inactivation takes place during calcium translocation across the mitochondrial membrane, which could account for limited release of Ca2+ from mitochondrial matrix through the pore itself as well as relatively narrow limits of the pore open state in comparison with time scale of complete cation release from depolarised mitochondria.     

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 calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

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.     

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 (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR 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.     

Ca2+ transport in mitochondria from yeast expressing recombinant aequorin

We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.     

Mitochondrial Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca(2+) and oxidants such as peroxynitrite (ONOO(-)). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca(2+) in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca(2+) was a critical factor in determining the impact of ONOO(-) on PTP. Specifically, addition of Ca(2+) alone, or ONOO(-) and then Ca(2+), elicited similar low levels of PTP opening, whereas ONOO(-) alone was ineffective. In contrast, addition of Ca(2+) and then ONOO(-) induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca(2+) reveals a molecular target that is now reactive with ONOO(-). As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO(-) alone or to Ca(2+) and then ONOO(-), and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca(2+) include dehydrogenases and CoA - containing enzymes. These data are discussed in the context of the role of mitochondria, Ca(2+), and ONOO(-) in apoptotic signaling.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.     

Protective role of transient pore openings in calcium handling by cardiac mitochondria

Long-lasting mitochondrial permeability transition pore (mPTP) openings damage mitochondria, but transient mPTP openings protect against chronic cardiac stress. To probe the mechanism, we subjected isolated cardiac mitochondria to gradual Ca(2+) loading, which, in the absence of BSA, induced long-lasting mPTP opening, causing matrix depolarization. However, with BSA present to mimic cytoplasmic fatty acid-binding proteins, the mitochondrial population remained polarized and functional, even after matrix Ca(2+) release caused an extramitochondrial free [Ca(2+)] increase to >10 μM, unless mPTP openings were inhibited. These findings could be explained by asynchronous transient mPTP openings allowing individual mitochondria to depolarize long enough to flush accumulated matrix Ca(2+) and then to repolarize rapidly after pore closure. Because subsequent matrix Ca(2+) reuptake via the Ca(2+) uniporter is estimated to be >100-fold slower than matrix Ca(2+) release via mPTP, only a tiny fraction of mitochondria (<1%) are depolarized at any given time. Our results show that transient mPTP openings allow cardiac mitochondria to defend themselves collectively against elevated cytoplasmic Ca(2+) levels as long as respiratory chain activity is able to balance proton influx with proton pumping. We found that transient mPTP openings also stimulated reactive oxygen species production, which may engage reactive oxygen species-dependent cardioprotective signaling.