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. 2012 Jan;12(1):120–125. doi: 10.1016/j.mito.2011.04.006

The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore

Pinadda Varanyuwatana 1, Andrew P Halestrap 1,⁎
PMCID: PMC3281194  PMID: 21586347

Abstract

Phosphate activation of the mitochondrial permeability transition pore (MPTP) opening is well-documented and could involve the phosphate carrier (PiC) that we have proposed is the pore's cyclophilin-D binding component. However, others have reported that following CyP-D ablation Pi inhibits MPTP opening while cyclosporine-A (CsA) inhibits MPTP opening only when Pi is present. Here we demonstrate that Pi activates MPTP opening under all energised and de-energised conditions tested while CsA inhibits pore opening whether or not Pi is present. Using siRNA in HeLa cells we could reduce PiC expression by 65–80% but this inhibited neither mitochondrial calcium accumulation nor MPTP opening.

Abbreviations: ANT, adenine nucleotide translocase; BKA, bongkrekic acid; CAT, carboxyatractyloside; CRC, calcium retention capacity; CsA, cyclosporine A; CyP-D, cyclophilin-D; PEG, poly(ethylene glycol); IMM, inner mitochondrial membrane; MPT, mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; NTA, nitrilotriacetic acid; OMM, outer mitochondrial membrane; PiC, mitochondrial phosphate carrier; PPIase, peptidyl-prolyl cis-trans isomerase; ROS, reactive oxygen species; SfA, sanglifehrin A; VDAC, voltage activated anion channel

Keywords: Permeability transition pore, Heart, Reperfusion injury, Phosphate, Calcium

1. Introduction

The mitochondrial permeability transition pore (MPTP) is a non-selective pore in the inner mitochondrial membrane (IMM), permeable to all solutes < 1.5 kDa. It opens in response to high matrix calcium concentrations but the sensitivity to [Ca2+] is greatly enhanced by oxidative stress, phosphate and adenine nucleotide depletion (Bernardi et al., 2006; Crompton, 1999; Halestrap, 2009). These conditions occur when cells are stressed such as occurs during reperfusion following a period of ischemia, and it is now widely accepted that MPTP opening is a critical mediator of necrotic cell death under these circumstances (Basso et al., 2008; Crompton, 2000; Di Lisa & Bernardi, 2009; Halestrap, 2010; Halestrap & Pasdois, 2009). Indeed, inhibition of MPTP opening, either pharmacological by MPTP inhibitors such as cyclosporine A (CsA) and sanglifehrin A (SfA) or indirectly by decreasing oxidative stress and calcium overload (e.g. by ischemic preconditioning) provide strong protection against reperfusion injury (Bernardi et al., 2006; Halestrap, 2010; Halestrap et al., 2004).

1.1. The molecular composition of the MPTP

Despite many years of research in several laboratories the detailed molecular mechanism of the MPTP remains uncertain although some proteins have been shown to play important roles. Thus, inhibition of MPTP opening by CsA and SfA implicated the matrix peptidyl-prolyl cis-trans isomerase activity of cyclophilin D (CypD), and this was subsequently confirmed through the use of CyP-D knockout mice (see (Azzolin et al., 2010; Halestrap, 2009)). However, genetic or pharmacological ablation of CyP-D does not abolish MPTP opening but rather decreases its sensitivity to [Ca2+]. This implies that CyP-D plays a facilitating rather than an essential role in MPTP opening. A role of the adenine nucleotide translocase (ANT) was implicated by the inhibitory effects of adenine nucleotides and bongkrekic acid, a ligand of the ANT that traps the ANT in the “m” conformation, and the activating effect of carboxyatractyloside (CAT) that traps the ANT in the “c” conformation (see (Halestrap & Brenner, 2003; Klingenberg, 2008)). These data were taken as evidence that the ANT might be the pore forming component of the MPTP, but subsequently the use of mitochondria from ANT knockout mice cast doubt on this conclusion. Thus mitochondria from these mice still displayed Ca-induced MPTP opening that was enhanced by oxidative stress, but pore opening was less sensitive to [Ca2+] and was no longer sensitive to adenine nucleotides and CAT (Kokoszka et al., 2004). These data can be interpreted in two ways. Either the ANT may play only a regulatory role or the ANT might be able to form the pore but in its absence other proteins could take over this function.

1.2. The role of the mitochondrial phosphate carrier in MPTP formation

Recent work performed in this laboratory has provided strong evidence that the mitochondrial phosphate carrier (PiC) may play a key role in MPTP formation (Leung et al., 2008). Thus we were able to demonstrate CsA-sensitive binding of CyP-D to the PiC whilst the modification of thiol groups on the PiC by oxidative stress and phenylarsine oxide (PAO) correlated with MPTP opening. Furthermore, the sensitivity of MPTP opening to inhibition by N-ethylmaleimide and ubiquinone 0 (UQ0) matched their ability to inhibit phosphate transport into mitochondria. UQ0 also prevented PAO from activating pore opening and inhibited the binding of the PiC to immobilised PAO. In the light of these data we suggested that the PiC might be a key component of the MPTP that undergoes a calcium-induced conformational change to induce pore formation. This conformational change might be enhanced by an interaction of the PiC with the “c” but not the “m” conformation of the ANT (Halestrap, 2009, 2010; Leung et al., 2008). We further proposed that binding of Pi to the PiC might provide an explanation for the ability of Pi to activate pore opening (Leung et al., 2008). Such activation of the MPTP by Pi in energised mitochondria has been well documented (Al-Nasser & Crompton, 1986; Chalmers & Nicholls, 2003; Crompton et al., 1988; Crompton & Costi, 1988; Roos et al., 1980), and we demonstrated a similar effect in de-energised mitochondria (Halestrap & Davidson, 1990).

However, more recent data from Bernardi's laboratory has suggested that the effects of Pi may be more complex (Basso et al., 2008; Di Lisa & Bernardi, 2009). To measure MPTP opening, these authors assayed the calcium retention capacity (CRC) of energised liver mitochondria from wild-type and CyP-D knockout mice in the presence or absence of CsA and phosphate. This assay determines the amount of calcium that must be taken up by the mitochondria before the MPTP opens and then releases the accumulated calcium. Since an anion is required to support energised calcium uptake into the mitochondria under these conditions, when phosphate was omitted, the phosphate analogues arsenate (Asi) or vanadate were added in its place. It was found that in the absence of Pi, ablation of CyP-D genetically (CyP-D knockdown) or pharmacologically (CsA addition) did not lead to an observable increase in CRC whereas in the presence of Pi it did. The authors interpreted these data as demonstrating that Pi actually inhibits (rather than activates) pore opening but that this effect is overcome by the presence of CyP-D, accounting for the protective effects of CsA in the presence of Pi. Since this effect of Pi was not shared by arsenate or vanadate, which are also substrates for the PiC, their data would argue against a role for the PiC in the MPTP. However, we have presented extensive data to show that under de-energised conditions in KSCN medium, the MPTP is inhibited by CsA in the absence of Pi (Griffiths & Halestrap, 1991; Halestrap et al., 1997; Halestrap & Davidson, 1990), which would argue against a requirement for Pi to demonstrate CsA inhibition of the MPTP. In this paper we confirm that Pi is not required to demonstrate CsA-sensitive pore opening in either energised or de-energised mitochondria incubated in standard KCl media. We also explore the use of siRNA knockdown to provide further evidence for a role for the PiC in the MPTP.

2. Materials and methods

2.1. Preparation of mitochondria

Mitochondria were prepared from livers of 250 g male Wistar rats following homogenisation in sucrose isolation buffer (300 mM sucrose, 10 mM Tris–HCl, 2 mM EGTA, pH 7.4) in a Dounce Potter homoginizer and purified by Percoll® density-gradient centrifugation as in Halestrap and Davidson (1990).

2.2. Measurement of MPTP opening in de-energised mitochondria

2.2.1. De-energised swelling

This was determined by following the decrease in light scattering (monitored as A520) as described previously (Halestrap et al., 1997; Halestrap and Davidson 1990). Mitochondria were incubated at 25 °C and 1 mg/ml in de-energised assay buffers containing 150 mM KCl or KSCN, 20 mM MOPS, 10 mM TRIS, 2 mM nitrilotriacetic acid (NTA), 0.5 μM rotenone, 0.5 μM antimycin A, 2 μM A23187, and when required either 20 mM Pi or 20 mM Asi. Swelling of mitochondria was initiated with 50 μM free [Ca2+] and A520 was monitored continuously in a spectrophotometer with computerised data acquisition.

2.2.1. Shrinkage assay

This technique was employed to determine the sensitivity of MPTP to [Ca2+] and [Pi] and was carried out as previously described (Connern and Halestrap 1994; Connern and Halestrap 1996). Mitochondria were incubated for 20 min at 30 °C and 2 mg/ml under standard de-energised KSCN buffer (as above) but without added NTA or A23187 and with addition of 1 mM CaCl2. Any residual swelling was terminated by an addition of 1.2 mM EGTA, which also resealed swollen mitochondria. The resulting swollen mitochondria were collected by centrifugation at 12,000 ×g for 10 min and resuspended at 2 mg/ml in either de-energised KCl or KSCN buffer without added NTA or Ca2+ ionophore. In order to ensure equilibrium of matrix with the new buffer, the swollen mitochondria were incubated again at 30 °C supplemented with 1 mM CaCl2. After 3 min, 1.2 mM EGTA was added to reseal the mitochondria before centrifugation at 12,000 ×g for 10 min. The swollen mitochondria were resuspended at 30 mg/ml in either de-energised KSCN or KCl buffer containing 2 mM NTA and 2 μM A23187.

The extent of MPTP opening in these pre-swollen mitochondria was determined by addition of poly(ethylene glycol), PEG 2000, to induce shrinkage. Initially 2 mg of swollen mitochondria was added to 3 ml of assay buffer containing the required free [Ca2+] and [Pi] or [Asi]. Free [Ca2+] was calculated as described in Rutter and Denton (1988) assuming the same binding constant of Ca2+ to Asi as to Pi. Our own measurements with Fura-6F (see below) suggested that this was a reasonable assumption. Shrinkage was initiated after exactly 1 min of incubation by a rapid addition of 0.5 ml 50% (w/v) PEG (to give a final PEG concentration of 7% w/v) and continuously monitored (10 data points per second) as an increase in A520.

2.3. Determination of MPTP opening in energised mitochondria

Simultaneous measurement of extramitochondrial [Ca2+] and mitochondrial membrane potential was performed using Fura-6F (Molecular Probe, F-15178) and Rhodamine-123 (Molecular Probe, R22420) in a multiwavelength fluorimeter (Cairn Instruments). Excitation was at 340 and 380 nm for Fura-6F and 490 nm for Rhodamine-123 with 90° fluorescence emission detected by a photomultiplier using a 520 nm bandpass filter. A second photomultiplier detected 90° light scattering at 490 nm. Excitation filters were contained in a spinning wheel rotating continuously at 24 Hz. Liver mitochondria (1 mg/ml) were incubated at 30 °C within a stirred cuvette containing 3 ml assay buffer containing 120 mM KCl, 10 mM MOPS, 5 mM L-glutamate, 2 mM L-malate, 20 μM EGTA, 1 μM Fura-6F, 100 nM Rhodamine-123 and either 1 mM Pi or 1 mM Asi, pH 7.2. Additions of Ca2+ were made as required through an injection port.

2.4. siRNA-knockdown of the PiC and assay of MPTP opening in HeLa cells

HeLa cells were cultured as described previously (Ullah et al., 2006). The siRNA used against the human PiC was 5′-CUGGCGCACAUCACUAUAUdTdT-3′ and was obtained from Sigma Gynosis who also provided an appropriate scramble siRNA to act as a control. A variety of transfection techniques were tested to establish the optimal conditions to give knockdown of the PiC without causing cell death. These included several different lipid-based reagents and the Amaxa Cell line Nucleofector®. We found that transfection using 75 pmoles of siRNA in 12 μl Dharmafect-1 agent (Dharmacon) and culturing for 72 h gave the best reduction in PiC expression as determined by Western blotting. Expression of the PiC and CyP-D (loading control) were determined in cell extracts using Western blotting as described previously (Leung et al., 2008). For determination of the sensitivity of the MPTP to [Ca2+] cells were harvested using trypsin, washed in PBS and permeabilised with digitonin (10 mg per 106 cells). After 12 min of incubation on ice, the cells were washed once in PBS before resuspending in assay buffer (120 mM KCl, 10 mM MOPS, 5 mM L-glutamate, 2 mM L-malate, 1 mM Pi, 20 μM EGTA, 1 μM Fura-6F and 100 nM Rhodamine-123, pH 7.2).

3. Results

3.1. CsA-sensitive MPTP opening does not require phosphate in energised mitochondria

In order to measure MPTP opening in energised liver mitochondria we have developed a technique to monitor mitochondrial swelling (light scattering) simultaneously with membrane potential (Rhodamine-123 fluorescence) and extra-mitochondrial [Ca2+] (Fura-6F). This is described more fully under Materials and methods (Section 2.3). In Fig. 1 panel A we compare the effects of 1 mM phosphate (Pi) and arsenate (Asi) on the response of mitochondria to sequential additions of Ca2+ in the presence and absence of 1 μM CsA. Experiments were also performed in the absence of either anion, but these led to impaired calcium uptake and MPTP-independent depolarisation as described by others (Basso et al., 2008; Chalmers & Nicholls, 2003; Nicholls, 1978) and thus were not employed further. Our data show that in the absence of CsA, the first few additions of calcium led to transient increases of extra-mitochondrial [Ca2+] as the added Ca2+ was rapidly taken up by the mitochondria. In the presence of 1 mM Pi, on addition of the 8th aliquot of 10 μM [Ca2+] a rapid loss of membrane potential and release of calcium were observed coincident with a rapid decrease in light scattering indicative of MPTP opening. In the presence of CsA 10 additions of 20 μM [Ca2+] followed by 4 additions of 40 μM [Ca2+] were required before MPTP opening was observed. When the experiment was repeated in the presence of 1 mM Asi in place of Pi, MPTP opening was observed after 6 and 11 additions of 10 μM [Ca2+] in the absence and presence of CsA respectively. These data show that more calcium additions were required to open the MPTP when Pi was used to support uptake of calcium than when Asi was used, but that sensitivity to CsA was apparent in both cases.

Fig. 1.

Fig. 1

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CsA-sensitive MPTP opening does not require phosphate. In panel A energised liver mitochondria were incubated in KCl medium containing 20 mM Pi or Asi with or without 1 μM CsA as indicated. Fura-6F and Rhodamine 123 fluorescence were continuously monitored together with light scattering and additions of CaCl2 made as indicated. In panel B mitochondria were incubated in de-energised KCl medium supplemented with 1 mM Pi or Asi with or without 1 μM CsA as indicated, and MPTP opening was determined following addition of 50 μM (free) Ca2+ by monitoring swelling (decrease in A520). Panel C shows the PEG-induced shrinkage of pre-swollen mitochondria incubated in KCl medium containing 60 μM free [Ca2+] in the presence or absence of 1 mM Pi or Asi with or without 1 μM CsA as indicated. In panel D mean data (± S.E.M.; n = 3) are presented for the sensitivity of MPTP opening to [Ca2+] determined by the PEG-shrinkage assay in KCl medium supplemented with either or both 5 mM Pi and 1 μM CsA as indicated. In order to normalise data between different batches of mitochondria rates of shrinking are expressed as a percentage of the rate in the presence of 60 μM Ca2+ and no phosphate. In panel E mean data (± S.E.M.; n = 3) are presented for the sensitivity of MPTP opening to Pi or Asi determined by the PEG-shrinkage assay in KCl medium at 60 μM [Ca2+]free. Further details of all methods used are given under “Materials and methods” (Section 2). Control (Con) refers to KCl medium in the absence of Pi or Asi.

3.2. Phosphate activates MPTP opening in de-energised mitochondria

Studying MPTP opening in energised mitochondria may be the most appropriate conditions to mimic the situation in vivo, but it is not ideal for studying the mechanism of the MPTP. This is because under energised conditions parameters such as membrane potential, ATP/ADP ratio and rates of calcium influx and efflux can all influence MPTP opening and thus an intervention that affects any of these parameters will also modulate MPTP opening indirectly (Halestrap, 2009). To circumvent this problem we usually work under de-energised conditions in the presence of the calcium ionophore, A23187, to ensure equilibration of [Ca2+] across the IMM. Under these conditions both Pi and Asi were found to sensitise the MPTP to [Ca2+] with Pi being more effective than Asi, and here too CsA inhibited MPTP opening whether or not Asi or Pi were present (Fig. 1 panel B).

In order to study the effects of Pi, Asi and CsA on the calcium-sensitivity of the MPTP in more detail we used the shrinkage assay. Here, mitochondria are pre-swollen by opening of the MPTP with high [Ca2+] before resealing by calcium chelation with EGTA and washing to remove released matrix components. These mitochondria can be induced to shrink upon addition of polyethylene glycol (PEG), and the rate of shrinkage indicates the extent of MPTP opening. This technique has the advantage that it releases matrix adenine nucleotides and other factors that may themselves influence the Ca-sensitivity of MPTP opening (Halestrap et al., 1997). The data of Fig. 1 panel C confirm that Pi increases the sensitivity of the MPTP to [Ca2+] (i.e. shifts the Ca-activation curve to the left) whilst CsA decreases the Ca-sensitivity of the MPTP, shifting the curve to the right. For comparison, we also performed parallel experiments with mitochondria incubated in the KSCN medium that we have routinely used in the past (Halestrap et al., 1997). These data (not shown) were similar to those observed in KCl medium in the presence of 5 mM Pi. In Fig. 1 panel D we show data for the concentration dependence of Pi and Asi activation of MPTP opening at fixed [Ca2+] (60 μM). As with the swelling data, at all concentrations employed, Pi was more effective than Asi at inducing MPTP opening.

3.3. The effects of siRNA knockdown of the PiC on the sensitivity of MPTP opening to [Ca2+]

Our previous data have implicated the PiC as the CyP-D binding component of the MPTP (Leung et al., 2008) and thus the PiC would seem to be a likely target for the effects of Pi and Asi. In order to confirm such a role for the PiC it would be desirable to genetically ablate the protein as has been done for the ANT and CyP-D using knockout mice (see (Forte & Bernardi, 2005)). However, phosphate transport across the inner membrane is essential for ATP production by oxidative phosphorylation and for the transport of other essential metabolites into mitochondria (Palmieri, 2004) which makes it unlikely that PiC knockout mice will be viable. As an alternative approach, we have used siRNA to knockdown the PiC in HeLa cells and assayed MPTP opening using mitochondrial light scattering, calcium retention and membrane potential measurements with Fura-6F and Rhodamine 123 following digitonin permeabilization of the cells. Western blotting revealed that 72 h after transfection with siRNA the reductions in PiC expression were between 65 and 80%, while longer incubations caused cell death which we assumed to reflect sufficient loss of PiC to compromise cell metabolism. We compared the [Ca2+]-sensitivity of MPTP opening in mitochondria of HeLa cells transfected with PiC siRNA with control cells transfected with scrambled siRNA. Typical data are presented in Fig. 2 for cells treated with scrambled siRNA (Panel A) and PiC siRNA (Panel B). It should be noted that the amounts of calcium added to induce MPTP opening are much smaller than for liver mitochondria because of the low number of mitochondria present in the permeabilized cells. Nevertheless, it is clear that MPTP opening could be detected after 8 or 9 calcium additions as indicated by the loss of membrane potential, release of Ca2+ and decrease in light scattering. The data show that there was no measurable difference between the cells treated with scrambled siRNA and PiC siRNA, despite the latter giving a decrease in PiC expression of about 65% (Fig. 2 panel C). These data demonstrate that knockdown of the PiC by 65–80% was not sufficient to impair either mitochondrial calcium uptake or MPTP opening. As a loading control in these experiments we used CyP-D, another MPTP component, and this showed no change in the cells transfected with PiC siRNA.

Fig. 2.

Fig. 2

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MPTP opening in mitochondria of HeLa cell subject to PiC knockdown. After 72 h of transfection with scrambled (panel A) or PiC siRNA (panel B), cells were harvested, washed and permeabilised with digitonin for measurement of MPTP opening. This was performed under energised conditions using Fura-6F and Rhodamine 123 fluorescence together with light scattering. Additions of CaCl2 were made as indicated. Panel C shows the expression of the PiC in cells transfects with scramble (Scr) or PiC siRNA as determined by Western blotting. CyP-D was used as a mitochondrial marker to confirm equivalent loading. Scanning the film revealed that the PiC knockdown was about 65%. Further details of all methods used are given under “Materials and methods” (Section 2).

4. Discussion

4.1. Phosphate activates MPTP opening under all conditions and is not required for CsA inhibition

The data we present in this paper show that under de-energised conditions Pi is an activator of the MPTP and its presence is not required to demonstrate the inhibitory effect of CsA on MPTP opening (Fig. 1 panel B and C). Thus our data do not support the proposal of Bernadi and colleagues (Basso et al., 2008) that Pi is an inhibitor of the MPTP and that this inhibitory effect is overcome by CyP-D in a CsA sensitive manner. It is possible that the effects of Pi are different between the de-energised conditions routinely used by us and the energised conditions used by Bernardi et al. However, when we reproduced the conditions of Bernardi et al. using energised conditions and the calcium retention assay (Basso et al., 2008) we were still able to detect an inhibitory effect of CsA on MPTP opening whether or not Pi was present as the anion accumulated with calcium (Fig. 1 panel A). Our data are in agreement with those of Bernardi et al. in one important respect; more calcium must be added to open the MPTP under energised conditions in the presence of Pi than Asi (Fig. 1 panel A). However, in our hands this is true whether or not CsA is present. By contrast, under de-energised conditions the sensitivity of the MPTP to [Ca2+] was greater in the presence of Pi than Asi (Fig. 1 panels B and D), suggesting that under these conditions Pi is a better activator of pore opening than Asi.

The reason for the opposite effects of Pi and Asi in energised and de-energised mitochondria may well reflect the greater ability of Pi to bind matrix calcium under energised conditions. There is evidence that calcium accumulated under energised conditions forms some ill-defined complex with Pi (Chalmers & Nicholls, 2003) which might not be shared by Asi. Importantly, when Chalmers and Nicholls investigated the relationship between MPTP opening and matrix [Ca2+]free in the presence of 1 and 5 mM Pi, they observed that the MPTP opened at much lower matrix [Ca2+]free at 5 mM Pi than at 1 mM Pi consistent with the activation of MPTP opening by phosphate we describe here (Chalmers & Nicholls, 2003).

4.2. Confirmation of a role for the PiC by siRNA knockdown may be difficult

In view of our published data on the possible involvement of the PiC in MPTP formation (Leung et al., 2008) it would be possible to explain the ability of Pi and Asi to activate MPTP opening by their binding to the PiC and inducing a conformation that favours the open state of the pore. The best way to confirm this would be through genetic ablation of the PiC, but our attempts to do this using siRNA reported here demonstrate the problems associated with such an approach. First, we found that the maximum knockdown we could achieve was 65–80% after 72 h. In our hands attempts to increase knockdown further led to cell death. In the cells with the highest attainable knockdown it would appear that there was still sufficient PiC to maintain maximal rates of calcium uptake without any detectable decrease in MPTP opening (Fig. 2). It should be noted that the siRNA we used will knock down both of the two PiC splice variants found in human mitochondria; PiC-A is present in heart and skeletal muscle and PiC-B is ubiquitous (Dolce et al., 1994; Dolce et al., 1996; Fiermonte et al., 1998). Our C-terminal antibody would detect both isoforms (predicted molecular masses of 34,896 and 34,760) which may explain why the PiC appears as a doublet in the Western blot (Fig. 2C) with both bands being reduced to a similar extent by the siRNA. Of course the lack of effect of PiC knockdown on MPTP opening may reflect a lack of involvement of the PiC in the MPTP formation. However, it is equally possible that the remaining 20–30% of the PiC is sufficient to give maximal MPTP opening. If we were able to produce much higher knockdown of the PiC without cell death it might be possible to distinguish between these possibilities. However, in the absence of the PiC, Pi will not enter the mitochondria to facilitate calcium accumulation. Thus it would not be possible to study MPTP opening using the fluorescent assay of CRC and membrane potential collapse routinely used in permeabilized cells. Rather, it will be necessary to develop a light scattering assay under de-energised conditions that could be used on the very small quantities of mitochondria that can be obtained from cultured cells.

Acknowledgements

This work was supported by a Programme Grant from the British Heart Foundation (RG/08/001/24717) and an Overseas Research Scholarship from the University of Bristol to PV.

References

  1. Al-Nasser I., Crompton M. The reversible Ca2+-induced permeabilisation of rat liver mitochondria. Biochem. J. 1986;239:19–29. doi: 10.1042/bj2390019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Azzolin L., von Stockum S., Basso E., Petronilli V., Forte M.A., Bernardi P. The mitochondrial permeability transition from yeast to mammals. FEBS Lett. 2010;584:2504–2509. doi: 10.1016/j.febslet.2010.04.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Basso E., Petronilli V., Forte M.A., Bernardi P. Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation. J. Biol. Chem. 2008;283:26307–26311. doi: 10.1074/jbc.C800132200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernardi P., Krauskopf A., Basso E., Petronilli V., BlalchyDyson E., DiLisa F., Forte M.A. The mitochondrial permeability transition from in vitro artifact to disease target. FEBS J. 2006;273:2077–2099. doi: 10.1111/j.1742-4658.2006.05213.x. [DOI] [PubMed] [Google Scholar]
  5. Chalmers S., Nicholls D.G. The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria. J. Biol. Chem. 2003;278:19062–19070. doi: 10.1074/jbc.M212661200. [DOI] [PubMed] [Google Scholar]
  6. Connern C.P., Halestrap A.P. Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel. Biochem. J. 1994;302:321–324. doi: 10.1042/bj3020321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connern C.P., Halestrap A.P. Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to [Ca2+] Biochemistry. 1996;35:8172–8180. doi: 10.1021/bi9525177. [DOI] [PubMed] [Google Scholar]
  8. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 1999;341:233–249. [PMC free article] [PubMed] [Google Scholar]
  9. Crompton M. Mitochondrial intermembrane junctional complexes and their role in cell death. J. Physiol. 2000;529:11–21. doi: 10.1111/j.1469-7793.2000.00011.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crompton M., Costi A. Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. A potential mechanism for mitochondrial dysfunction during cellular Ca2+ overload. Eur. J. Biochem. 1988;178:489–501. doi: 10.1111/j.1432-1033.1988.tb14475.x. [DOI] [PubMed] [Google Scholar]
  11. Crompton M., Ellinger H., Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 1988;255:357–360. [PMC free article] [PubMed] [Google Scholar]
  12. Di Lisa F., Bernardi P. A CaPful of mechanisms regulating the mitochondrial permeability transition. J. Mol. Cell. Cardiol. 2009;46:775–780. doi: 10.1016/j.yjmcc.2009.03.006. [DOI] [PubMed] [Google Scholar]
  13. Dolce V., Iacobazzi V., Palmieri F., Walker J.E. The sequences of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms. J. Biol. Chem. 1994;269:10451–10460. [PubMed] [Google Scholar]
  14. Dolce V., Fiermonte G., Palmieri F. Tissue-specific expression of the two isoforms of the mitochondrial phosphate carrier in bovine tissues. FEBS Lett. 1996;399:95–98. doi: 10.1016/s0014-5793(96)01294-x. [DOI] [PubMed] [Google Scholar]
  15. Fiermonte G., Dolce V., Palmieri F. Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria. J. Biol. Chem. 1998;273:22782–22787. doi: 10.1074/jbc.273.35.22782. [DOI] [PubMed] [Google Scholar]
  16. Forte M., Bernardi P. Genetic dissection of the permeability transition pore. J. Bioenerg. Biomembr. 2005;37:121–128. doi: 10.1007/s10863-005-6565-9. [DOI] [PubMed] [Google Scholar]
  17. Griffiths E.J., Halestrap A.P. Further evidence that cyclosporin-A protects mitochondria from calcium overload by inhibiting a matrix peptidyl-prolyl cis-trans isomerase — implications for the immunosuppressive and toxic effects of cyclosporin. Biochem. J. 1991;274:611–614. doi: 10.1042/bj2740611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halestrap A.P. What is the mitochondrial permeability transition pore? J. Mol. Cell. Cardiol. 2009;46:821–831. doi: 10.1016/j.yjmcc.2009.02.021. [DOI] [PubMed] [Google Scholar]
  19. Halestrap A.P. A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection. Biochem. Soc. Trans. 2010;38:841–860. doi: 10.1042/BST0380841. [DOI] [PubMed] [Google Scholar]
  20. Halestrap A.P., Brenner C. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 2003;10:1507–1525. doi: 10.2174/0929867033457278. [DOI] [PubMed] [Google Scholar]
  21. Halestrap A.P., Davidson A.M. Inhibition of Ca2+-induced large amplitude swelling of liver and heart mitochondria by Cyclosporin A is probably caused by the inhibitor binding to mitochondrial matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem. J. 1990;268:153–160. doi: 10.1042/bj2680153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Halestrap A.P., Pasdois P. The role of the mitochondrial permeability transition pore in heart disease. Biochim. Biophys. Acta. 2009;1787:1402–1415. doi: 10.1016/j.bbabio.2008.12.017. [DOI] [PubMed] [Google Scholar]
  23. Halestrap A.P., Woodfield K.Y., Connern C.P. Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J. Biol. Chem. 1997;272:3346–3354. doi: 10.1074/jbc.272.6.3346. [DOI] [PubMed] [Google Scholar]
  24. Halestrap A.P., Clarke S.J., Javadov S.A. Mitochondrial permeability transition pore opening during myocardial reperfusion — a target for cardioprotection. Cardiovasc. Res. 2004;61:372–385. doi: 10.1016/S0008-6363(03)00533-9. [DOI] [PubMed] [Google Scholar]
  25. Klingenberg M. The ADP and ATP transport in mitochondria and its carrier. Biochim. Biophys. Acta. 2008;1778:1978–2021. doi: 10.1016/j.bbamem.2008.04.011. [DOI] [PubMed] [Google Scholar]
  26. Kokoszka J.E., Waymire K.G., Levy S.E., Sligh J.E., Cal J.Y., Jones D.P., MacGregor G.R., Wallace D.C. The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature. 2004;427:461–465. doi: 10.1038/nature02229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leung A.W.C., Varanyuwatana P., Halestrap A.P. The mitochondrial phosphate carrier interacts with cyclophilin d and may play a key role in the permeability transition. J. Biol. Chem. 2008;283:26312–26323. doi: 10.1074/jbc.M805235200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nicholls D.G. The regulation of extramitochondrial free calcium concentration by rat liver mitochondria. Biochem. J. 1978;174:463–474. doi: 10.1042/bj1760463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Palmieri F. The mitochondrial transporter family (SLC25): physiological and pathological implications. Pflugers Arch. 2004;447:689–709. doi: 10.1007/s00424-003-1099-7. [DOI] [PubMed] [Google Scholar]
  30. Roos I., Crompton M., Carafoli E. The role of inorganic phosphate in the release of Ca2+ from rat-liver mitochondria. Eur. J. Biochem. 1980;110:319–325. doi: 10.1111/j.1432-1033.1980.tb04870.x. [DOI] [PubMed] [Google Scholar]
  31. Rutter G.A., Denton R.M. Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilised rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD + ratios. Biochem. J. 1988;252:181–189. doi: 10.1042/bj2520181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ullah M.S., Davies A.J., Halestrap A.P. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1 alpha-dependent mechanism. J. Biol. Chem. 2006;281:9030–9037. doi: 10.1074/jbc.M511397200. [DOI] [PubMed] [Google Scholar]

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