The permeability transition pore complex: another view

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1 Biochimie 84 (2002) The permeability transition pore complex: another view Andrew P. Halestrap *, Gavin P. McStay, Samantha J. Clarke Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK Received 14 August 2001; accepted 20 December 2001 Abstract Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kda, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca 2+ ], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca 2+ or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys 56 and Cys 159 ) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys 159 modification whilst oxidation of Cys 56 increases CyP-D binding to the ANT, probably at Pro Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Adenine nucleotide translocase; Cyclophilin-D; Calcium; Oxidative stress; Mitochondrial swelling; Reconstitution; MPTP mechanism; Voltage activated anion channel 1. Introduction The primary role of mitochondria in healthy cells is the provision of ATP to support normal cell function and, until recently, this had been the major focus of research into mitochondrial function. However, in recent years the central role of mitochondria in apoptotic and necrotic cell death has become apparent, and a major player in this arena is the Abbreviations: ANT, adenine nucleotide translocase; CAT, carboxyatractyloside; BKA, bongkrekic acid; CsA, cyclosporin A; CyP, cyclophilin; IMM, inner mitochondrial membrane; MPT, mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; NEM, N-ethylmaleimide; CuP, copper phenanthroline; PAO, phenylarsine oxide; VDAC, voltage activated anion channel * Corresponding author. Tel.: ; fax: address: A.Halestrap@Bristol.ac.uk (A.P. Halestrap). mitochondrial permeability transition (MPT). The MPT refers to the massive swelling and depolarisation of mitochondria that occurs under some conditions, most notably as a result of calcium overload and oxidative stress. The cause of the MPT is the opening of a non-specific pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (MPTP). A major consequence of the MPT is the uncoupling of oxidative phosphorylation. This will prevent mitochondria making ATP by oxidative phosphorylation and also, by reversal of the mitochondrial proton-translocating ATPase, actively stimulate the hydrolysis of ATP produced by glycolysis. Unrestrained, this will inevitably lead the cell towards necrotic death. Indeed pore opening has been shown to be a critical event in the necrotic cell death that occurs when tissues are subjected to insults such as reperfusion injury and chemical toxins (see [1,2]) Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII:S (02)

2 154 A.P. Halestrap et al. / Biochimie 84 (2002) In addition to its role in necrosis, transient MPTP opening may be involved in apoptosis through the release of cytochrome c and other pro-apoptotic molecules. These then activate the caspase cascade that sets apoptosis in motion [1 3]. However, it seems probable that only if subsequent MPTP closure occurs will ATP levels be maintained, ensuring that cell death continues down an apoptotic rather than a necrotic pathway [4]. Nevertheless, mitochondria play an important role in apoptosis even in the absence of the MPT since release of pro-apoptotic factors such as cytochrome c, apoptosis inducing factor (AIF) and Smac/Diablo from the inter-membrane space of mitochondria may occur through changes in the outer membrane permeability. These are induced by pro-apoptotic proteins such as BAX and Bid [3,5]. The main emphasis of the work performed in this laboratory over the past 12 years has been to elucidate the molecular mechanism of the MPTP and its role in reperfusion injury of the ischaemic heart. In this review we will restrict our attention to the former and the reader is referred elsewhere for a review of the extensive work from this and other laboratories on the role of the MPTP in reperfusion injury in the heart and brain, and the development of protective strategies that may be applicable to clinical situations [1,6,7]. 2. The MPT in historical context The phenomenon of the MPT was first described several decades ago as a massive swelling of mitochondria that accompanies calcium overload. It was initially thought to be a result of non-specific damage to the inner mitochondrial membrane (IMM) by phospholipases [8]. However, pioneering studies, initiated by Hunter and Haworth in the late seventies [9,10] and confirmed 7 years later by Martin Crompton and colleagues [11], implied that the pore is a unique molecular entity that allows the passage of any molecule of < 1500 daltons across the IMM, and can be rapidly closed by chelation of calcium. Because the MPTP allows rapid passage of protons, its opening is accompanied by depolarisation of the mitochondria and uncoupling of oxidative phosphorylation. In addition, the equilibration of all small solutes across the IMM leaves behind high concentrations of proteins in the matrix and these exert a colloidal osmotic pressure that is responsible for the extensive swelling of mitochondria associated with the MPT [1,12]. Our own interest in the MPTP arose from an investigation into the regulation of ATP production by hormones since these studies led us to investigate the physiological role of changes in mitochondrial volume. A brief summary of this work follows because it provided us with important clues about the mechanism of the MPTP. 3. Mitochondrial swelling MPTP-dependent and independent mechanisms Studies on the hormonal regulation of hepatic metabolism in this and other laboratories during the seventies and early eighties had demonstrated that stimulation of respiration and oxidative phosphorylation are critical events [13]. It became apparent that these effects involve an increase in mitochondrial [Ca 2+ ] and activation of the three calciumsensitive mitochondrial dehydrogenases, pyruvate dehydrogenase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. The resulting increase in matrix NADH/NAD + stimulates respiration leading to an increase in proton motive force (pmf) and ATP production [14]. However, the rise in NADH is only transient and is followed by a decline to basal levels even though rates of respiration, ATP production and the pmf remain elevated. This re-oxidation of NADH is associated with a calcium-mediated increase in mitochondrial matrix volume of 20 40% that could be detected by a small decrease in light scattering as well as by direct measurement of volume with 3 H 2 O and [ 14 C]- sucrose. Such small increases in matrix volume cause a stimulation of NADH oxidation by increasing the rate of electron flow from Complex 1 into ubiquinone, an effect that can be mimicked by 1 5 nm valinomycin, a potassium ionophore. This provides an explanation of why the hormonally induced increase in mitochondrial NADH/NAD + is only transient whilst the pmf and ATP production rates remain stimulated (see [13,15]). These results have recently been confirmed at the single cell level (see [16]). The calcium-dependent increase in matrix volume is caused by entry of K + ions through a potassium channel, driven by the membrane potential, with proton-driven co-transport of the phosphate anion providing charge compensation. Osmotically obliged water follows, causing the relatively modest (20 40%) swelling of the matrix (c.f. the massive swelling that accompanies the permeability transition). Extensive studies on this calcium activated potassium channel led to the conclusion that the protein responsible is the adenine nucleotide translocase (ANT) an integral inner membrane protein whose normal role is the translocation of ATP and ADP across the IMM. This is converted into a channel when the ATP/ADP binding sites are not occupied by nucleotides, an event which happens rarely under resting conditions. However, when matrix [Ca 2+ ] is elevated, pyrophosphate (PPi) accumulates in the matrix and may transiently displace adenine nucleotides from some ANT molecules. The increase in [PPi] is caused by the powerful inhibition of pyrophosphatase by micromolar [Ca 2+ ], but elevating matrix [PPi] independently of Ca 2+, for example by provision of butyrate, can also cause a modest increase in mitochondrial matrix volume [15]. It was during these studies that Martin Crompton reported that the permeability transition could be specifically inhibited by cyclosporin A (CsA) [17]. This key observation led us to consider whether the physiological changes in

3 A.P. Halestrap et al. / Biochimie 84 (2002) mitochondrial volume we had been measuring in response to hormones might be related to the massive swelling associated with the permeability transition. The lack of an effect of CsA on the physiological response clearly demonstrates that this is not the case [18] but introduced the laboratory to the permeability transition pore and the possibility that under some conditions, the ANT can act as a channel as well as a transporter [19]. 4. Experimental protocols used to study the permeability transition in isolated mitochondria The MPT can be studied in isolated mitochondria by a variety of methods (see [1,2]). These include: Measuring the swelling of mitochondria by monitoring the associated decrease in light scattering. As mitochondria swell their refractive index changes and they scatter less light. This can be detected as a decrease in light absorbance measured with a spectrophotometer. To avoid any complications that changes in the redox state of respiratory chain components might cause, the wavelength of the incident light should be at the isosbestic point for the cytochromes (520 nm). Following the loss of mitochondrial membrane potential using fluorescent dyes or a TPMP + electrode. A disadvantage of this technique is that uncoupling does not necessarily mean that the MPTP has opened, and it is important to confirm this by showing inhibition of the response by CsA. Measurement of the release of accumulated calcium with a calcium-sensitive dye or electrode. The same criticisms apply to this technique as to measurements of the membrane potential. Measurement of [ 14 C] sucrose permeation into mitochondria. This is a very reliable technique that has been used primarily by Crompton s laboratory (see [2]), but requires specialised apparatus. In this laboratory we routinely employ light scattering to investigate the mechanism of the MPTP and prefer to use de-energised mitochondria (no respiratory substrates present and the electron transport chain inhibited with rotenone and antimycin A). A calcium ionophore is also added to ensure that calcium equilibrates across the inner mitochondrial membrane. Although such conditions are far removed from the in vivo situation, they minimise the possibility of indirect effects that added reagents may have on the permeability transition. For example, any molecule that depolarises the mitochondria following calcium accumulation will cause opening of the MPTP, whilst any inhibitor of calcium entry into mitochondria will prevent the MPTP. Yet in neither case is the effect of the reagent directly on the MPTP mechanism. We have also found that more consistent results can be obtained using mitochondria that are left on ice for h before use. The reason for this is that, following their preparation, mitochondria lose an increasing proportion of their adenine nucleotides during the first few hours of storage on ice. This causes the sensitivity of the MPTP towards calcium to change substantially over the time period of an experiment, whereas leaving mitochondria for 18 h before use minimises this effect. Another trick that ensures reproducible results is to perform the light scattering experiment in an iso-osmotic KSCN buffer. The reason behind this choice of buffer is that it is often used for determining mitochondrial potassium fluxes, but it so happens that its mild chaotropic action sensitises the MPT to calcium. The mechanism underlying this effect will be discussed below. A variation of the light scattering technique, first introduced by Hunter and Haworth [9,10], is to pre-swell the mitochondria by opening the MPTP with excess calcium and then induce shrinkage of the mitochondria in the presence of polyethylene glycol (PEG) of about 4 kda. This molecule is too large to permeate the MPTP, and thus exerts an osmotic pressure on the matrix even when the MPTP is open. The advantage of this technique is that the composition of the matrix can be varied at will once the MPTP is open in the pre-swollen state, and thus matrix effectors such as [ADP] can be studied very readily. The extent of pore opening is reflected in the rate at which the light scattering decreases upon PEG addition. 5. Characteristics of the MPTP The primary trigger for opening of the MPTP is a rise in matrix [Ca 2+ ], but the concentration required is highly dependent on the prevailing conditions (see [1,8,20]). Several factors are known to greatly enhance the sensitivity of the pore to [Ca 2+ ], of which the most potent and relevant to the cellular setting are oxidative stress, adenine nucleotide depletion, increased inorganic phosphate concentrations and mitochondrial depolarisation. Under energised conditions (but not de-energised conditions), the pore is also sensitive to ligand-induced conformational changes of the ANT being activated by carboxyatractyloside (CAT) and inhibited by bongkrekic acid (BKA). Low ph (< 7.0) is a potent inhibitor of the MPTP probably as a result of protons competing with Ca 2+ for its binding at the trigger site [9,21,22]. The specificity of this site for calcium appears to be absolute and other divalent cations such as Sr 2+,Mn 2+,Ba 2+ and Mg 2+ act as inhibitors [9,22]. In addition to the calcium-specific trigger site on the matrix surface, there is another inhibitory divalent cation binding site on the external surface of the inner membrane with a K i for Mg 2+ of about 0.3 mm [23]. An important factor that must be considered in relation to the effects of Mg 2+ on the MPTP is that ATP binds magnesium tightly (K d 10 4 at ph 7.0) and the Mg ATP complex does not bind to the ANT. Thus, energisation of mitochondria, which in its own right protects against MPTP opening, will also decrease free [ATP] and [Mg 2+ ] that inhibit pore opening and the overall effect may be hard to

4 156 A.P. Halestrap et al. / Biochimie 84 (2002) predict. More recently, it has been shown that ubiquinone analogues can act either as activators or inhibitors of the MPTP [24]. A much fuller list of known effectors of the MPTP may be found elsewhere [8]. A well-documented feature of the permeability transition is that for any individual mitochondria it is an all or none phenomenon. That is, the mitochondria are either normal or massively swollen with no intermediate state being apparent (see [20]). However, within a population, individual mitochondria possess different sensitivities to inducers of the MPTP. Thus, the magnitude of the decrease in light scattering observed in a suspension of mitochondria reflects the number of mitochondria in the population that have undergone the permeability transition rather than a progressive and concerted increase in volume of all the mitochondria. The stochastic nature of the MPT can readily be explained since once a single pore opens, rapid proton entry and adenine nucleotide loss will induce more pores to open, leading to a full transition. Other mitochondria within the population will then take up the calcium that is released by those that have undergone the transition and some of these will now undergo the transition themselves. This will lead to a progressive opening of all the mitochondria that is reflected in the progressive decrease in light scattering [20]. 6. The molecular mechanism of the MPTP 6.1. The role of cyclophilin D The discovery that opening of the MPTP could be inhibited specifically by sub-micromolar concentrations of the immunosuppressive drug, cyclosporin A (CsA) [17] provided the impetus for our own research into the molecular mechanism of the MPTP. The immunosuppressive effect of CsA involves its binding to a cytosolic protein called cyclophilin-a (CyP-A) [25] and this led us to investigate the role of a matrix CsA binding protein (cyclophilin). Cyclophilins exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity, and catalyse the interconversion between cis and trans conformations of peptide bonds adjacent to proline residues [25]. As such they are ideally suited for enabling the conformational change in a membrane protein that would be required to induce formation of a pore. We were able to demonstrate that the mitochondrial matrix contains such a CsA-sensitive PPIase [19] and that the K 0.5 values of several CsA analogues for the inhibition of this PPIase activity correlated with their potency as inhibitors of pore opening [19,26,27]. This led us to propose in 1990 that the MPTP was formed from an interaction between the adenine nucleotide translocase (ANT) and mitochondrial CyP [19] as discussed further below. Subsequently we were able to purify, clone and sequence the CsA-sensitive mitochondrial PPIase [28,29] and hence confirm that it was a unique cyclophilin, the rat equivalent of the human CyP-3 gene product [30] and distinct from cytosolic cyclophylin A. Now known as cyclophilin D (CyP-D), this nuclear encoded protein has a mitochondrial targeting presequence that is cleaved after translocation of the protein into the matrix. Cleavage occurs at one of two points leading to mature proteins of about 17.6 kda (minor product) and 18.6 kda (major product) [28]. Subsequently, Crompton and colleagues also purified and sequenced mitochondrial CyP-D [31] and confirmed that the protein can be imported into the mitochondria before cleavage of its presequence [32]. Northern blots demonstrate that mrna for CyP-D is present in rat muscle, heart, liver, kidney and brain and is of identical size (1.5 kb) in all tissues [29]. This makes it unlikely that there are differently spliced tissue-specific isoforms that might account for the different sensitivities of mitochondria from various tissues. For a soluble protein such as CyP-D to play a role in the opening of the MPTP it must interact with a membrane protein and induce a conformational change when triggered by Ca 2+. Indeed, it could be envisaged that factors known to sensitize mitochondria to pore opening, such as oxidative stress, might do so by enhancing binding of CyP-D to its target membrane protein. In support of this we demonstrated that oxidative stress induced by t-butyl hydroperoxide (TBH) or diamide treatment increase CyP-D binding to the inner mitochondrial membrane in parallel with increasing the sensitivity of pore opening to [Ca 2+ ] [33,34]. Interestingly, in order to show this effect consistently, the mitochondrial membranes had to be prepared in iso-osmotic KSCN medium to stabilise the complex between CyP-D and its membrane target protein. Stabilisation could also be achieved by the addition of low concentrations of guanidinium hydrochloride, implying that it is the chaotropic property of KSCN that is responsible for its stabilising effects [34]. This suggests that the CyP-D forms a complex with the target protein, inducing a conformational change that exposes more of the protein surface to the aqueous medium. Such an effect might be predicted for the formation of a channel. Another factor that was shown to enhance both CyP-D binding and MPTP opening in response to [Ca 2+ ]is an increase in matrix volume [34]. The cause of this is unknown although it may reflect the accessibility of CyP-D to its target membrane binding site. Whatever the mechanism, one significant consequence is that once the permeability transition has occurred and mitochondria have swollen, more CyP-D is likely to become bound making reversal of the MPT more difficult. In our experiments, no matter what conditions were used to stimulate binding of CyP to the IMM, the process was almost totally prevented by CsA. However, in our hands other known modulators of the MPTP such as matrix [Ca 2+ ], [ADP], ph or membrane potential were without effect [33 35]. In contrast, Bernardi and colleagues have demonstrated an inhibitory effect of low ph on CyP-D binding to sub-mitochondrial particles. The effect was blocked by the histidine reagent diethylpyrocarbonate which also blocks the inhibitory effect of ph on the MPTP [36]. These experiments were performed in low

5 A.P. Halestrap et al. / Biochimie 84 (2002) ionic strength media where a large number of other matrix proteins also remained bound to the membrane at low ph, and thus it is possible that the effect of ph was on non-specific binding of CyP-D to charged groups on the phospholipids or membrane proteins. Crompton and colleagues used similar conditions when labelling the membrane bound CyP-D with photoactivatable CsA derivatives [37]. Their data suggested that Ca 2+ might enhance and ADP diminish CyP binding under such conditions, but our own experiments did not confirm this [34,38]. Although it appears to be well established that CsA blocks pore opening by binding to CyP-D, there are data to indicate that CyP binding may not be required for the MPTP to open at very high matrix [Ca 2+ ]. Thus, Novgorodov et al [39] and Crompton and Andreeva [40], using different techniques, have shown that at high matrix [Ca 2+ ] inhibition of pore opening by CsA is overcome. We have confirmed this in both heart mitochondria [26] and liver mitochondria [33 35]. Yet under the same conditions, CsA is able to prevent almost totally the binding of CyP-D to the inner mitochondrial membrane [33,35]. In addition, studies on the megachannel of patched clamped mitochondria have shown that an inhibitory effect of CsA is overcome at higher [Ca 2+ ] [41]. Although it is dangerous to assume that the megachannel studied electrophysiologically is necessarily a manifestation of the same molecular entity as the MPTP [42], it does appear that CyP-D binding is not absolutely essential for the MPTP to open, but may rather sensitise the process to [Ca 2+ ]. This is not unreasonable if it is assumed that pore opening involves a cis-trans isomerisation around a proline residue that causes a conformational change of the protein. This process could occur independently of CyP-D binding, but be greatly enhanced by the bound CyP-D The role of the adenine nucleotide translocase Indirect evidence An involvement of the ANT in pore opening was first proposed by Hunter and Haworth [10] and more convincing evidence provided by LeQuoc and LeQuoc [43] and ourselves [19,35]. The early evidence was based largely upon the observation that in energised mitochondria, any reagent such as CAT that stabilised the c conformation of the ANT, sensitised the MPT to [Ca 2+ ], whilst any reagent such as BKA that stabilised the m conformation of the ANT, made the MPT less sensitive to [Ca 2+ ]. In passing it should be noted that many workers have used CAT and BKA as specific activators and inhibitors of the MPTP in cultured cell models of apoptosis. In doing so they fail to recognise that the primary effect of these reagents will be to prevent ATP/ADP exchange across the mitochondrial inner membrane, with profound effects on cellular metabolism irrespective of any effect on the MPT. It should also be noted that CAT and its less potent analogue atractyloside bind to the ANT with very high affinity (K d < 1 µm), and thus their use at µm in some published experiments may well induce non-specific effects quite unrelated to their binding to the ANT. Matrix ADP is another important modulator of pore opening that acts by decreasing the sensitivity of the calcium trigger site to [Ca 2+ ]. There are two ADP binding sites with K i values of about 1 and 25 µm. The high affinity site is blocked by the inhibitor CAT and is therefore thought to be associated with the ANT [9,10,35,44,45]. The identity of the second site is less clear but may well be an extramitochondrial binding site for adenine nucleotides on the ANT. We tested the ability of a range of nucleotides to inhibit the MPT, and found that apart from ADP, only ATP and deoxy-adp inhibit with K 0.5 values 500 and 20 times greater than ADP respectively. This correlates with their affinity for the matrix binding site of the ANT [35]. Bernardi and colleagues have provided strong evidence that the MPTP is voltage-regulated, being activated as the membrane potential becomes less negative (see [20,46]). We have suggested that the membrane potential is sensed by the ANT itself through an effect on adenine nucleotide binding. This is possible because the ANT catalyses an electrogenic exchange of ATP 4 for ADP 3 with a mechanism that may well involve a potential driven conformational change altering the affinity of the ANT for adenine nucleotides on either side of the membrane [47,48]. In support of this hypothesis, we have demonstrated that in mitochondria depleted of adenine nucleotides by pyrophosphate treatment, not only is the MPT much more sensitive to [Ca 2+ ], but it is also no longer voltage sensitive [19,27]. Adenine nucleotide binding is antagonised by oxidative stress induced by reagents such as t-butylhydroperoxide (TBH) or diamide and also by thiol reagents such as phenylarsine oxide (PAO), a powerful activator of the MPT [35,49]. PAO has the greatest effect of the reagents tested, raising the K 0.5 for ADP inhibition of the MPT to > 500 µm [35]. We have shown that this effect is accompanied by covalent modification of the ANT [35] which may explain why PAO is a more potent stimulus of the MPT than diamide or TBH, and yet has a smaller effect on CyP-D binding [34]. Oxidative stress and PAO also shift the voltage dependence of the MPT, allowing the pore to open at more negative potentials. This is exactly what would be predicted if thiol modification inhibits adenine nucleotide binding to the matrix surface of the ANT whilst membrane potential enhances this binding CyP-D binds specifically to the ANT The data described above strongly supports a critical role for the ANT in the formation of the MPTP, as we originally proposed in 1990 [19], but falls short of proof. In order to confirm that CyP-D does bind specifically to the ANT under conditions favouring opening of the MPTP we overexpressed CyP-D as a fusion protein with glutathione-stransferase (GST) for use as a CyP-D affinity column. When solubilised IMMs were passed over this column and weakly binding proteins washed off, only one protein remained

6 158 A.P. Halestrap et al. / Biochimie 84 (2002) bound and this was the ANT. Binding was inhibited by pre-treatment of the GST CyP-D with CsA and enhanced when the inner membranes were prepared from mitochondria subjected to oxidative stress with diamide [38]. In similar studies, Crompton and colleagues found that both the voltage activated anion channel (VDAC, also known as porin) and the ANT bound tightly to the GST CyP column, but in contrast to our own studies they were unable to prevent binding with CsA [50]. These apparent discrepancies may be the result of major differences in the experimental protocol used in the two studies. Thus, Crompton et al. used unfractionated heart mitochondria whereas our own studies employed purified inner membranes from liver mitochondria. Since heart and liver mitochondria possess different isoforms of the ANT (mainly ANT1 and ANT2, respectively), it is possible that the relative affinities of these isoforms for CyP-D and VDAC are different. Another difference is that Crompton solubilised the mitochondria with the zwitterionic detergent CHAPS whilst in our own studies the IMMs were solubilised in the non-ionic detergent Triton-X Thiol groups on the ANT play a critical role in pore opening Bernardi and colleagues have provided data that suggest two distinct thiol groups are implicated in modulating MPTP activity [51,52]. One is sensitive to oxidation of glutathione, for example by TBH or diamide, and is protected by both monobromobimane and N-ethylmaleimide. The other responds to the redox state of matrix NAD(P), and is protected by N-ethylmaleimide but not monobromobimane. It is the latter site that accounts for the well documented stimulatory effect of oxidation of matrix NADH on the MPT, perhaps through the mediation of thioredoxin or lipoamide [53]. The ANT is known to have three cysteine residues that show differential reactivity towards various thiol reagents and oxidising agents in a conformation dependent manner [54 56]. These cysteines may well represent the thiol groups that regulate both CyP-D binding and the inhibitory effects of ADP and membrane potential on the MPT [35]. In support of this, eosine maleimide, that specifically attacks cysteine 159 of the ANT within the adenine nucleotide binding site [54,57], also abolishes the ability of ADP to inhibit the opening of the MPTP [35]. Furthermore, the ANT binds specifically to a phenylarsine oxide column [35] and this binding is prevented by pre-treatment with eosine maleimide or diamide (unpublished data of Gavin McStay and Andrew Halestrap). These data suggest that PAO and diamide treatment are likely to cross-link either Cys 159 with Cys 56 or Cys 256 with Cys 56. The latter has particular attractions because CyP-D is likely to bind to a proline residue in view of its peptidyl- prolyl cis-trans isomerase activity, and there is a proline residue, Pro 61, close to Cys 56 that we suggested in our original model might be the binding site for CyP-D [19]. Fig. 1. CyP-D inhibits dimeristion of the adenine nucleotide translocase by copper/o-phenanthroline in sub-mitochondrial particles. SMPs were treated with 400 µm copper/o-phenanthrolene (CuP) for 10 min at 0 C before (B) or after (A) of 2 µm GST CyP-D addition followed by sedimentation and through washing. Samples were subject to SDS PAGE under non-reducing conditions and xestern blotting with anti-ant or -CyP-D antibodies. This proline is absent in the three ANT isoforms found in yeast mitochondria which do not exhibit a CsA-sensitive MPTP [35]. Furthermore, a cross-link between Cys 159 and Cys 56 would explain why oxidative stress is such a potent activator of the pore since, by modifying both Cys 159 and Cys 56, it would affect both adenine nucleotide and CyP-D binding. Brenner and colleagues have also presented data to suggest an important role for Cys 56 in opening of the MPTP suggesting that cross-linking between two ANT molecules may be important [58]. However, we have shown that diamide treatment of mitochondria does not lead to the formation of Cys 56 cross-linked dimers of the ANT. Interestingly, we have shown that when such cross-linking is induced in sub-mitochondrial particles (SMPs) with copper phenanthroline (CuP) treatment, it can be inhibited by the addition of exogenous CyP-D as illustrated in the previously unpublished data of Fig. 1. This suggests that when CyP-D binds it protects Cys 56 from cross-linking which is consistent with our proposal that CyP-D binds to Pro 61. In other unpublished experiments we have used other thiol reagents to establish unequivocally which cysteine residue cross-links to Cys 159. N-ethylmaleimide (NEM) at low concentrations (50 µm) specifically attacks Cys 56 whilst CuP can cross-link two Cys 56 residues to produce an ANT dimmer [54]. We found that both diamide and 50 µm NEM prevented CuP induced dimerisation. These data suggest that diamide cross-links Cys 56 to Cys 159. By way of confirmation, 50 µm NEM was also found to prevent the diamide induced increases in CyP-D binding to submitochondrial particles and of ANT to a GST CyP-D affinity column. In contrast, we suggest that PAO cross-links

7 A.P. Halestrap et al. / Biochimie 84 (2002) Fig. 2. Location of cysteine residues in the ANT whose cross-linking modulates MPTP activity. Cys 256 to Cys 159 since binding of the ANT to a PAO-column is not inhibited by 50 µm NEM. Since PAO requires vicinal thiols for binding and Cys 56 is not involved (see above), this leaves only Cys 256 and Cys 159 to which PAO can bind. As would be predicted, ANT binding to the PAO column is greatly reduced by diamide or eosine maleimide treatment, both of which attack Cys 159. A scheme illustrating our current thinking on the role of thiol groups of the ANT in MPTP function is shown in Fig Other proteins may interact with the ANT to enhance or inhibit pore opening In recent years there have been several reports that a range of pro-apoptic proteins, including Bax and the viral protein R of the HIV-1 virus, and anti-apoptotic proteins including Bcl-2 and the viral protein vmia of the cytomegalovirus, interact directly with the ANT to exert their effects on apoptosis (see [59,60]). However, there are aspects of this work that need to be treated with caution. For example, although an inhibitory effect of Bcl-2 on the MPTP in situ has been widely reported (see [61,62]), there are data on isolated mitochondria that fail to support this view. Thus, it has been demonstrated that the MPT occurs in liver mitochondria from mice over-expressing Bcl-2 with the same sensitivity to inducers as mitochondria from control mice [63]. Another report suggests that Bcl-2 prevents the permeability transition by enabling the reduced pyridine nucleotides to be maintained during oxidative stress rather than through a direct effect on the MPTP mechanism [64]. The proposed interaction between BAX and the ANT was originally identified using a yeast two hybrid screen using BAX as bait, that pulled out a peptide containing residues of the ANT (containing a transmembrane helix and parts of both an extramitochondrial and intramitochondrial loop) [65]. There are, however, always concerns as to whether protein/protein interactions detected with yeast two hydrid screens are specific and occur in the physiological setting. This is especially true with membrane proteins since the transcription factor interactions that underlie the yeast two hybrid technique require both expressed proteins to be soluble in the cytosol, which is unlikely for membrane proteins. Thus, the interaction between a transmembrane helical region of the ANT and BAX could reflect no more than the natural tendency of the hydrophobic ANT sequence to pair up with another hydrophobic sequence in BAX. Such an interaction might produce a hydrophilic surface that keeps the proteins in solution whilst activating expression of the reporter gene. Nevertheless, some evidence for specificity of the BAX / ANT interaction is provided by binding competition studies that locate the region of the ANT to which Vpr and Bcl-2 bind as residues [66]. This sequence overlaps with that identified by the yeast two hybrid screen and is contained within an extramitochondrial loop, as might be expected for the binding of cytosolic proteins. However, there are also problems associated with the use of the reconstituted ANT in binding studies, since this process requires solubilisation of the ANT in detergent whilst maintaining its native state, followed by reconstitution and detergent removal. None of these procedures is straightfor

8 160 A.P. Halestrap et al. / Biochimie 84 (2002) ward as will be considered below. Despite these misgivings, it remains an attractive hypothesis that other proteins may target the ANT, the most abundant protein of the inner mitochondrial protein, and exert their effects on apoptosis or necrosis by modulating the MPT Other possible components of the MPTP It is important to recognise that the permeability transition is fundamentally an inner membrane phenomenon associated with uncoupling of the mitochondria, swelling of the matrix and unfolding of the inner membrane cristae. Indeed, the MPT can be observed in mitoplasts, mitochondria from which the outer membrane is removed, with identical properties to that observed with normal mitochondria [35]. This is not to deny that proteins of the outer membrane may be involved in the MPT, and it is certainly true that the MPT leads to outer membrane rupture. However, at the heart of the MPTP must be a channel across the inner membrane. Our data strongly suggest that the only inner membrane protein to which CyP-D binds is the ANT whereas the data of Crompton et al. [50] suggest that VDAC may also bind, perhaps through a secondary interaction with the ANT. This highlights an ongoing controversy in the literature as to the minimum configuration of the MPTP. Does the open pore consist only of the ANT and CyP-D as we originally proposed or are other components required such as VDAC and the peripheral benzodiazepine receptor (see [1 3]). These proteins were shown to co-purify with the ANT as a complex under some conditions [67] and are also thought to interact at contact sites, points of intimate contact between the inner and outer membranes [68]. It was these observations that led Zoratti and Szabo in their seminal review [41] to propose that VDAC and the peripheral benzodiazepine receptor might be integral components of the MPTP. Although this was only a hypothesis, and not based on experimental evidence, it was perceived as established fact by many of those working on apoptosis who were becoming aware of the critical role of mitochondria in this process. Since that time the groups of Kroemer and Brdizka have suggested that other proteins associated with the contact sites, such hexokinase and creatine kinase may also be involved in the MPTP [61]. Indeed fractions of detergent solubilised proteins containing these components, along with CyP-D, could be reconstituted into proteoliposomes to form a calcium activated, CsA-sensitive pore [58,69]. However, which of the components are essential to pore formation could not be established by such techniques. The evidence is perhaps most compelling for VDAC since Crompton and colleagues were able to reconstitute the ANT VDAC complex eluted from a GST CyP-D affinity column glutathione into proteoliposomes to form a calcium activated pore that was inhibited by CsA [50]. However, as recognised by Crompton et al., this does not demonstrate that VDAC is essential for MPTP formation, and the question remains as to the minimum configuration of the MPTP? Is VDAC an essential component or does it play a regulatory role? Are there other proteins that are essential for MPTP function or regulation? Can both the major ANT isoforms form the MPTP and with similar affinities for CyP-D and other regulatory proteins such as VDAC? The most powerful approach to answering these questions would be to reconstitute the active MPTP complex from purified components, but this has proved extremely difficult to do with any consistency Reconstitution of the MPTP Support for an involvement of the ANT in the formation of the MPTP had come from earlier studies in which it had been shown that thiol reagents were able to convert the reconstituted ANT from a specific antiporter to a nonspecific channel [70]. Subsequently it was shown that the reconstituted ANT can also form non-specific pores when exposed to high (millimolar) concentrations of calcium [71,72]. These pores differ from the MPTP in that they are not sensitive to CsA, but their permeability properties are similar. Initial reports of reconstitution of the MPTP from the laboratories of Brdiczka and Kroemer used crude fractions from whole homogenised brain rather than purified components and are thus hard to interpret (see [61]). Subsequently, Kroemer s group went on to use the purified, reconstituted ANT in studies on the MPTP (see [59], whilst Crompton s group reported reconstitution of CsA-sensitive pore activity using the CyP-D/ANT/VDAC complex isolated by glutathione elution from GST CyP-D affinity column [50]. However, in our view these experiments fail to take into account three major problems associated with any attempt to reconstitute the MPTP from its component proteins and thus make interpretation of the results obtained difficult. The first problem is that of solubilising the membrane proteins in a suitable detergent without denaturing them and maintaining their activity through purification. For example, it is well documented that unless stabilised by binding of a high affinity ligand such as BKA or atractyloside (ATR), the ANT rapidly denatures when solubilised in detergent [73]. It is significant that in laboratories specialising in measuring the activity of the reconstituted ANT, studies are performed at low temperature and over a millisecond time scale [48,73]. In contrast, recent studies using the reconstituted ANT for investigating the MPTP have confirmed ANT activity by measuring transport over 1 h at room temperature (see e.g. [74]). The second problem is the need to obtain sealed, detergent-free proteoliposomes, with the ANT in a defined orientation, preferably with the matrix surface facing out to allow binding of exogenous cyclophilin. A particular problem in many reported studies is the use of an inappropriate step for the removal of Triton-X100 following reconstitution of the solubilised proteins. The

9 A.P. Halestrap et al. / Biochimie 84 (2002) low critical micellar concentration and large micellar size of this detergent means that it cannot be properly removed from proteoliposomes by dilution and gel filtration [75]. Since even very low concentrations of Triton-X100 can exert profound effects on both the inhibitor sensitivity of VDAC [76] and on the general permeability of lipososomes [77], results obtained in this way must be treated with caution. The third problem is that a method must be found to assay pore opening in small amounts of reconstituted material. The published studies from other laboratories have used the release of a radioactive, fluorescent or chromogenic marker molecule entrapped within the proteoliposome. In many cases the release was measured after min at room temperature (e.g. [62,66] yet total release of the marker from a proteoliposome would be expected to occur almost instantaneously once the MPTP had opened. Furthermore, this approach does not allow continuous recording of pore activity to determine kinetics of activation and regulation since the release of the marker is an all or none phenomenon. Aware of the problems listed above, we have performed our own studies using rapidly purified ANT reconstituted into proteoliposomes in the presence and absence of CyP-D [78]. The most reliable published purifications of the ANT utilise high affinity ligands, such as BKA or ATR to stabilise the ANT during detergent solubilisation and subsequent purification [73]. Unfortunately, in our hands, CyP-D does not bind to the ATR ANT and BKA ANT complexes [38], and thus we cannot use this approach to maintain ANT stability. As an alternative, we have endeavoured to use a very rapid purification procedure for the ANT, using Triton- X100 solubilised heart mitochondria with hydroxyapatite chromatography followed by ion exchange FPLC [72] and immediate reconstitution of the pure protein fraction in proteoliposomes, using Biobeads to remove detergent. To assay the reconstituted MPTP we have developed a continuous spectrophotometric technique in which malate dehydrogenase is entrapped within the proteoliposomes and is only accessible to its substrates (NADH and oxaloacetate) when the pore opens. This is monitored by the decrease in A 340 as NADH is oxidised, and thus it is possible to determine the rate at which pores open as well as the fraction of pores that are open under any particular condition. We have used this technique to demonstrate that in the presence of recombinant CyP-D, the purified, reconstituted ANT does form CsA-sensitive pores at micromolar [Ca 2+ ](Fig. 3). These data are important because they confirm that the minimum configuration of the MPTP includes only the ANT and CyP-D, and any role for other proteins such as VDAC is likely to be regulatory rather than essential. However, we have not been able to use the reconstituted system to confirm this or to study detailed kinetics and regulation of the pore because the success rate for reconstitution of active MPTP has been < 10%. We have concentrated our efforts on Fig. 3. Reconstitution of the MPTP from purified ANT and CyP-D. The purified ANT was reconstituted into proteoliposomes containing entrapped malate dehydrogenease (MDH). Conversion of NADH to NAD + by the entrapped malate dehydrogenase is only possible when the MPTP opens. Ca 2+ and recombinant CyP-D were added externally and pore opening followed by the oxidation of NADH measured spectrophotometrically at 340 nm. Data are taken from [78] where further details may be found. establishing the reasons for this pore success rate and how it can be overcome. Recent data suggest that our standard purification procedure for the ANT causes substantial S-Scross-linking of Cys 56 to form ANT dimers that we know to be inactive in pore formation. The problem has been that when reagents such as dithiothreitol are used to maintain the reduced state of the ANT the purification procedure no longer works. 7. Genetic approaches to studying the MPTP It does not take much imagination to recognise that a powerful approach to elucidating the mechanisms of the MPTP would be a genetic approach in which proposed components of the MPTP are either knocked out totally or subjected to site-directed mutagenesis. Yeast would seem the organism of choice for such studies especially since it appears that their mitochondria do not display a conven

10 162 A.P. Halestrap et al. / Biochimie 84 (2002) tional CsA-sensitive MPT [35,79] and yeast ANT knockouts are available. Unfortunately, expressing active mammalian ANT isoforms in yeast mitochondria has proved extremely difficult but recent reports suggest that it can be done if the N-terminus of the yeast ANT is spliced onto the mammalian isoform [80]. In conjunction with the expression mammalian CyP-D this may provide an alternative approach to reconstitution in studying the molecular mechanism of the MPTP and has the advantage of being accessible to sitedirected mutagenesis. However, it must always be borne in mind that over-expression of an IMM protein may disrupt normal mitochondrial function and lead to cell death irrespective of whether or not it induces the formation of the MPTP. This may be the real basis of the report that overexpression of ANT-1 in mammalian cells can dominantly induce apoptosis [81] 8. A model of the MPTP that explains the mode of action of different modulators In Fig. 4 we summarise our current understanding of the mechanism of the MPTP, which represents a development of the model originally presented in 1990 [19], and which forms the basis of most models in the literature. We propose that CyP-D binds to the ANT on Pro 61 and that this binding is greatly enhanced when Cys 56 is cross-linked (e.g. by oxidative stress) to Cys 159. Yeast mitochondria lack both these residues and do not possess a CsA-sensitive MPTP [35,79]. We suggest that Ca 2+ binds to a site on the ANT itself, perhaps involving some of the many aspartate and glutamate residues on intramitochondrial loops of the ANT, but to date we have no information on the exact location. Binding of adenine nucleotides to the substrate binding site of the ANT greatly reduces the sensitivity of the pore to [Ca 2+ ], probably by masking the calcium binding site. Binding of calcium is thought to trigger the conformational change required to induce pore formation. This is likely to involve a cis-trans isomerisation of the peptide bond adjacent to Pro 61 that is facilitated by CyP-D binding but not totally dependent on it. This is consistent with the ability of high (mm) [Ca 2+ ] to convert the reconstituted to a pore in the absence of CyP-D [71]. Furthermore, at high calcium concentrations or upon adenine nucleotide depletion, the pore becomes insensitive to CsA [27,35]. Our model is able to provide a plausible explanation of the effects of most known regulators of the pore that may act to modulate binding of adenine nucleotides, calcium or CyP-D to their respective sites as summarised in Table 1. Thus, any intervention that reduces adenine nucleotide binding enhances pore opening. Factors acting this way include adenine nucleotide depletion, increased matrix phosphate or pyrophosphate concentrations (competes for the nucleotide binding site), and the conformational state of the ANT. The latter can be influenced by depolarisation (decreases the affinity of the matrix binding site for ADP) and specific ligands of the ANT such as carboxyatractyloside (decrease matrix ADP binding affinity) and BKA (increases matrix ADP binding affinity). Furthermore, modification of a specific thiol group on the ANT (Cys 159 ) either by oxidative stress or thiol reagents such as eosine maleimide or phenylarsine oxide, also decreases adenine nucleotide binding and can account for the ability of these agents to activate the pore [35]. Factors that enhance cyclophilin binding and hence increase the sensitivity of the MPTP to [Ca 2+ ] include oxidative stress (Cys 56 Cys 159 crosslinking), chaotropic agents and increased matrix volume. In contrast, CsA has the opposite effect by preventing CyP-D binding. Bernardi and colleagues have presented data to show that the effect of low ph involves a specific histidine residue and that this may modulate CyP binding to the inner mitochondrial membrane [36]. However, in our experiments we did not detect such an effect of low ph on CyP binding to either inner membranes [34] or to the purified ANT [38]. Rather, we suggest that protons (low ph), Mg 2+ and other divalent cations compete directly with Ca 2+ at the trigger site. The modes of action of two other potent inhibitors of the MPTP are not so clear. Trifluoperazine is a potent inhibitor of the MPT under energised but not de-energised conditions [35]. It was originally thought to act indirectly through inhibition of phospholipase A 2, preventing the accumulation of free fatty acids that are known to stimulate the MPTP, probably through interaction with the ANT. However, inhibition occurs even without changes in free fatty acid accumulation and is now thought to be mediated by an effect on surface membrane charge that changes the voltage sensitivity of the MPTP [82]. The mechanism of action of ubiquinone analogues as either as activators or inhibitors of the MPTP is also unclear [24,83]. Earlier work had shown that the probability of pore opening varied according to the rate of electron transfer through Complex 1 of the respiratory chain respiratory [83]. These data led Fontaine and colleagues to suggest that components of Complex 1 may be involved in the formation and/or regulation of the MPTP [24, 84]. However, the recent observation that the uncoupling proteins UCP1, UC2 and UCP3 require oxidised ubiquinone to function suggest an alternative mechanism [85, 86]. Since the UCPs are close relatives of ANT it would seem quite likely that a ubiquinone binding site may also exist on the ANT. 9. Conclusions Although falling short of definitive proof, the available evidence strongly supports a model for the MPTP in which the ANT and CyP-D are the key components, with other proteins in the outer mitochondrial membrane and cytosol (such as VDAC and Bcl-2) playing regulatory roles. However, urgently required are reproducible protocols for the reconstitution of the active MPTP from its pure components

11 A.P. Halestrap et al. / Biochimie 84 (2002) Fig. 4. The proposed mechanism of the mitochondrial permeability transition pore. The model shown includes a role for the ANT in physiological volume changes induced by calcium as well as in the formation of the MPTP at higher calcium concentrations, and is a development of that originally proposed in [19]. The sensitivity of the pore to calcium can be influenced by factors that bind directly to the Ca 2+ trigger site such as Mg 2+ and H + or that enhance CyP-D binding and displace adenine nucleotides from the ANT such as oxidative stress. The probable sites of action of the major known effectors of pore opening are shown in Table 1. Note that interactions of the ANT with outer membrane proteins such as VDAC (porin), the peripheral benzodiazipine receptor, Bcl2 family members and viral proteins may play a regulatory role. Table 1 Proposed sites of action of known effectors of the mitochondrial permeability transition. Further details may be found in the text Acting via Cyp-D binding a Acting via nucleotide binding a Acting directly on Ca 2+ binding Unknown mode of action Activators Vicinal thiol reagents (e.g. PAO) Oxidative stress to cross-link Cys 56 with Cys 159 of the ANT (e.g. TBH and Diamide) Increased matrix volume Chaotropic agents Thiol reagents attacking Cys 159 of ANT (e.g. PAO and eosine maleimide) Oxidative stress to cross-link Cys 56 with Cys 159 of the ANT (e.g. TBH and Diamide) C Conformation of ANT Adenine nucleotide depletion High matrix [Pi] and [PPi] High ph Some ubiquinone analogues (e.g. decyl-ubiquinone, ubiquinone 10) Inhibitors CsA and some analogues Membrane potential Low ph Some ubiquinone analogues (e.g. 2,5-dihydroxy-6-undecyl-1,4- benzoquinone) e.g. cyclosporin G, [MeAla 6 ] CsA and 4-methyl-val-CsA. M Conformation of ANT Mg 2+,Mn 2+,Sr 2+,Ba 2+ Trifluoperazine (may work via membrane surface charge) a Note that both CyP binding and ADP binding exert their effects through changes in the sensitivity of the MPT to [Ca 2+ ].