The mitochondrial permeability transition pore: a mystery solved?

Abstract

The mitochondrial permeability transition

The permeability transition (PT) defines an increase of mitochondrial inner membrane permeability to ions and solutes with molecular masses up to about 1500 Da leading to matrix swelling. The occurrence of swelling in isolated mitochondria, its strict dependence on matrix Ca²⁺, stimulation by Pi and fatty acids, and inhibition by Mg²⁺ and adenine nucleotides has been recognized very early (Raaflaub, 1953a,b; Brenner-Holzach and Raaflaub, 1954; Hunter and Ford, 1955; Tapley, 1956; Lehninger, 1959; Lehninger and Remmert, 1959; Wojtczak and Lehninger, 1961; Zborowski and Wojtczak, 1963; Azzi and Azzone, 1965; Azzone and Azzi, 1965; Chappell and Crofts, 1965; Crofts and Chappell, 1965); and its detrimental effects on energy conservation have been described before the emergence of chemiosmotic concepts (Mitchell, 1961, 2011). As a result, the PT has initially been considered an in vitro artifact of questionable pathophysiological relevance, although a few Authors did recognize its potential importance in pathophysiology (Rasola and Bernardi, 2007).

Pfeiffer and Coworkers suggested that the permeabilizing effect of Ca²⁺ had a physiological role in steroidogenesis. These Authors showed that Ca²⁺ induces a “transformation” of adrenal cortex mitochondria allowing diffusion of extramitochondrial pyridine nucleotides through the otherwise impermeable inner membrane, and that NADPH entering in this way supports the 11-β hydroxylation of deoxycorticosterone (Pfeiffer and Tchen, 1973, 1975; Pfeiffer et al., 1976). These findings match those of Vinogradov et al., who demonstrated the Ca²⁺-dependent release of matrix pyridine nucleotides in liver mitochondria (Vinogradov et al., 1972). The term “permeability transition” was introduced by Haworth and Hunter, who thoroughly characterized its basic features in heart mitochondria. These Authors provided the key insight that the PT was due to reversible opening of a proteinaceous pore in the inner mitochondrial membrane, the permeability transition pore (PTP), whose physiological role remained undefined (Hunter et al., 1976; Hunter and Haworth, 1979a,b; Haworth and Hunter, 1979).

Studies on the PT were not too popular at this time, a possible consequence of the general acceptance of the chemiosmotic hypothesis which had just been fully recognized with the award of the Nobel Prize to Peter Mitchell in 1978. As noted earlier (Bernardi, 1999) studies of mitochondrial ion transport were indeed carried out in the same laboratories involved in clarifying the mechanisms of energy conservation, and they tended to become tests of the predictions of the chemiosmotic theory. In retrospect it is not too surprising that the existence of a large pore in the inner membrane appeared to contradict the basic tenets of chemiosmosis. As a result, and with very few exceptions (see e.g., Beatrice et al., 1980; Coelho and Vercesi, 1980; Bernardi and Pietrobon, 1982; Lê-Quôc and Lê-Quôc, 1982, 1985; Siliprandi et al., 1983; Vercesi, 1984; Riley and Pfeiffer, 1985; Al Nasser and Crompton, 1986; Sokolove and Shinaberry, 1988) research in this area did not enjoy much popularity.

The discovery that in mammalian mitochondria the PT can be inhibited by submicromolar concentrations of cyclosporin (Cs) A (Fournier et al., 1987; Crompton et al., 1988; Broekemeier et al., 1989; Davidson and Halestrap, 1990) changed the field substantially. CsA inhibits the PTP after binding to matrix cyclophilin (CyP)D, a peptidyl-prolyl cis-trans isomerase (PPIase) whose enzymatic activity is blocked by CsA (Fischer et al., 1989; Takahashi et al., 1989) in the same range of concentrations inhibiting the pore (Connern and Halestrap, 1992, 1994; Nicolli et al., 1996; Woodfield et al., 1997). A second fundamental finding was the demonstration that mitochondria possess ion channels that can be studied by electrophysiology (Sorgato et al., 1987). This seminal study was soon followed by the demonstration that the inner mitochondrial membrane is endowed with a high-conductance (≈1–1.3 nS) channel, the “mitochondrial megachannel” (MMC) (Kinnally et al., 1989; Petronilli et al., 1989). The MMC is inhibited by CsA (Szabó and Zoratti, 1991) and possesses all the key regulatory features of the PTP (Bernardi et al., 1992; Szabó et al., 1992) leaving little doubt that the PTP and the MMC are two aspects of the same molecular entity (Szabó and Zoratti, 1992). Electrophysiology has greatly contributed to our understanding of the MMC–PTP, and to acceptance of the pore theory of the PT (Zoratti et al., 2005).

Involvement of the PT in cell death was hypothesized 25 years ago (Crompton and Costi, 1988). Early support was obtained in hepatocytes subjected to oxidative stress (Broekemeier et al., 1992; Imberti et al., 1992), anoxia (Pastorino et al., 1993), or treatment with ATP (Zoeteweij et al., 1993); and in cardiomyocytes (Duchen et al., 1993) and isolated hearts (Griffiths and Halestrap, 1995) exposed to ischemia followed by reperfusion. The exponential increase in experimental papers dealing with the PT as an effector mechanism of cell death, however, only followed the demonstration that in the course of apoptosis cytochrome c is released into the cytosol (Liu et al., 1996) together with apoptosis-inducing factor (Susin et al., 1996) and a set of other proteins involved in the effector phase of apoptosis (Du et al., 2000; Ekert et al., 2000; Hegde et al., 2001; Li et al., 2001).

The molecular basis of the PT is still the matter of conjectures (Siemen and Ziemer, 2013). The various models and working hypotheses proposed over the years had to cope with the lack of selectivity for the permeating species, the strict requirement for matrix Ca²⁺, which has recently been established also for yeast mitochondria (Yamada et al., 2009), the stimulation by oxidants, the existence of a vast number of “inducers” without common structural features, and of a more limited but still substantial number of inhibitors (Gunter and Pfeiffer, 1990).

Many suspects, no culprits

Cyclophilin D and F_oF₁ ATP synthase

CyPs are ubiquitous, conserved proteins possessing PPIase activity (Fischer et al., 1989; Takahashi et al., 1989) sharing a common domain of about 109 amino acids, the CyP-like domain (Wang and Heitman, 2005). In man 16 unique CyPs have been found, the prototype being cytosolic CyPA (Wang and Heitman, 2005). After binding CsA the PPIase activity is inhibited (Borel et al., 1977) and the CsA/CyPA complex binds to and inhibits the cytosolic phosphatase calcineurin (Liu et al., 1991), causing immunosuppression (Clipstone and Crabtree, 1992; Walsh et al., 1992).

CyPD is the mitochondrial CyP isoform in mammals (Connern and Halestrap, 1992; Nicolli et al., 1996; Woodfield et al., 1997), see Giorgio et al. (2010) for a review. Much on its role in PTP regulation has been learned by genetic ablation of the Ppif gene (which encodes for CyPD). CyPD is the mitochondrial receptor for CsA and modulates the PTP but is not a structural pore component (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005). As discussed elsewhere (Bernardi et al., 2006), the effect of CsA on the PTP is best described as “desensitization” in the sense that the PTP becomes more resistant to opening by Ca²⁺ and Pi. It should be stressed that pore opening readily takes place for Ca²⁺-Pi loads that are about twice those required in wild-type mitochondria. This indicates that Ppif^−/− (CyPD-null) mitochondria are not null for the PTP. This consideration is important for the interpretation of results obtained with CsA because (similarly to the absence of CyPD) CsA can desensitize but not block the PTP; thus, lack of sensitivity to CsA does not necessarily mean that the PTP is not involved in the event being studied. Furthermore, expression of CyPD is modulated, e.g., by muscle denervation (Csukly et al., 2006), and only CyPD-expressing mitochondria respond to CsA (Li et al., 2012). No endogenous ligands of CyPD mimicking the effects of CsA are known, but other PTP (de)sensitizers might act by favoring CyPD association or dissociation, as documented for acidic pH and increasing ionic strength (Nicolli et al., 1996). Furthermore, CyPD phosphorylation (Rasola et al., 2010), acetylation (Shulga and Pastorino, 2010), and nitrosylation (Kohr et al., 2011; Nguyen et al., 2011) affect the propensity of the PTP to open (Rasola and Bernardi, 2011). Not surprisingly CyPD may interact with many proteins including Hsp90 and its related molecule TRAP-1 (Kang et al., 2007); Bcl-2 (Eliseev et al., 2009); ERK-2/GSK-3 (Rasola et al., 2010); possibly p53 (Vaseva et al., 2012), but see (Karch and Molkentin, 2012); and the F_OF₁ ATP synthase (Giorgio et al., 2009). The latter interaction turned out to be the key for identification of the PTP.

The F_OF₁ ATP synthase (or complex V) is the rotary enzyme that synthesizes the vast majority of ATP in respiring cells (Rees et al., 2009). It is formed by the catalytic F₁, the membrane-bound proton-translocating F_O, and the lateral stalk linking F₁ and F_O (Strauss et al., 2008; Thomas et al., 2008; Rees et al., 2009; Baker et al., 2012; Davies et al., 2012) (Figure ​(Figure1).1). The lateral stalk acts as a stator preventing the α₃β₃ subcomplex of F₁ from rotating with subunits γ, δ, and ε and the ring of F_O-subunits c (Rees et al., 2009). The F_OF₁ ATP synthase complex associates to form dimers, which are considered to be the “building blocks” of long rows of oligomers that promote cristae formation and may optimize the enzyme's catalytic performance (Campanella et al., 2008; Strauss et al., 2008; Thomas et al., 2008; Rees et al., 2009; Baker et al., 2012; Davies et al., 2012). The angle formed by the dimers seems to display a large variability, and dimers are believed to represent the physiological unit of complex V in the inner membrane (Thomas et al., 2008).

Open in a separate window

Figure 1

Schematic representation of F_OF₁ ATP synthase dimers. F₁ (dark blue), F_O (green and light blue), and stalk subunits (red) are illustrated based on recent structural studies (Strauss et al., 2008; Baker et al., 2012; Davies et al., 2012).

We found that CyPD binds the lateral stalk of complex V in an apparent ratio of 1:1:1:1 with the OSCP, b and d subunits (Giorgio et al., 2009). CyPD binding requires Pi and results in partial inhibition of ATP synthase activity, while CsA displaces CyPD resulting in enzyme reactivation (Giorgio et al., 2009). The stimulatory effect of CsA on enzyme catalysis is lost in Ppif^−/− mitochondria, proving that it is mediated by CyPD, while assembly of the ATP synthase is not affected by CyPD ablation (Giorgio et al., 2009). In spite of the striking analogies with PTP regulation (Pi dependence, CsA sensitivity) whether the interactions of CyPD with complex V are relevant for the PT remained unknown until very recently.

Dimers of F_oF₁ ATP synthase form channels indistinguishable from the MMC–PTP

We have defined the binding site of CyPD on ATP synthase to the OSCP subunit, and shown that the interaction is electrostatic in nature. Modeling of surface potentials and isopotential curves suggested that CyPD interacts with OSCP in a region overlapping with helices 3 and 4, and this turned out to be a key insight (Giorgio et al., 2013). Indeed, this is also the binding site of Bz-423, a well-characterized inhibitor of the F_OF₁ ATP synthase (Johnson et al., 2005; Stelzer et al., 2010). If the CyPD interactions are important for the PT, it was logical to expect that Bz-423 should act as a PTP inducer, and this was the case. Bz-423 sensitized the PTP to Ca²⁺, and the inducing effect was blunted by Pi at concentrations that increase CyPD binding to OSCP (Giorgio et al., 2013). Consistent with an involvement of complex V in PTP formation, decreased levels of OSCP did affect the Ca²⁺ dependence of the pore. Indeed, mitochondria took up Ca²⁺ normally when ATP was used as the energy source, but the threshold Ca²⁺ load required for PTP opening was halved, which is the first molecular evidence that OSCP affects the probability of pore opening. Consistent with a key role of the ATP synthase, the Ca²⁺ affinity of the PTP was also affected by enzyme catalysis in the sense that ATP-hydrolyzing mitochondria required twice the Ca²⁺ load of ATP-synthesizing organelles, an effect as large as that of CsA (Giorgio et al., 2013).

Direct evidence that the PTP forms from the F_OF₁ ATP synthase was obtained by incorporating dimers purified by blue native electrophoresis into azolectin bilayers, and by measuring the passage of currents after application of a voltage difference. Addition of Bz-423 in the presence of Ca²⁺ triggered opening of high-conductance channels that were blocked by Mg²⁺/ADP and by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a non hydrolyzable ATP analog). Monomers of ATP synthase were devoid of channel activity in spite of the same overall subunit composition as the dimers (Tomasetig et al., 2002; Giorgio et al., 2013). The characteristics of the reconstituted pore closely matched those of MMC–PTP (Petronilli et al., 1989; Szabó and Zoratti, 1991; Szabó et al., 1992). Maximal chord conductance was between 1.0 and 1.3 nS in symmetrical KCl 150 mM, and various subconductance states were observed. Like the MMC of Ppif^−/− mitochondria (De Marchi et al., 2006), and in keeping with the lack of CyPD in the preparations, the channel was insensitive to CsA. Ca²⁺ and Pi, which sensitize the PTP even in the absence of CyPD, sufficed to induce PTP currents when dimers were prepared in the presence of 10 mM Pi. Channel openings could not be elicited by atractyloside and, once elicited by Bz-423, were still observed in the presence of bongkrekic acid. These findings are consistent with the absence of ANT in the preparations, which also totally lacked VDAC (Giorgio et al., 2013).

Bz-423 was characterized as an apoptosis-inducing agent acting through mitochondria (Blatt et al., 2002) and OSCP was identified as its target through the unbiased screening of a human phage display library (Johnson et al., 2005). The selectivity of action of Bz-423 on OSCP, its ability to trigger channel activity of ATP synthase dimers, and the lack of activity of monomer preparations strongly argues against the possibility that the currents observed by Giorgio et al. (2013) are due to unidentified contaminating proteins. The PTP-inducing effect of Bz-423 and CyPD (which both act through OSCP on top of the lateral stalk in the matrix) must be indirect, as it affects the permeability properties of the inner membrane. We assume that the PTP forms at the membrane interface between two adjacent F_O sectors, which could also accommodate the effects of fatty acids (Lehninger and Remmert, 1959; Wojtczak and Lehninger, 1961; Bernardi et al., 2002). Matrix Ca²⁺ has an essential permissive role in PTP formation, and we have suggested that accessibility of Ca²⁺ to the metal binding sites of the catalytic F₁ sector is influenced by OSCP. This subunit could indeed affect the affinity of the metal binding sites of ATP synthase and thus determine the ease with which matrix Ca²⁺ can replace Mg²⁺ causing PTP opening. Our working hypothesis is that OSCP as such is a “negative” modulator, whose effect can be counteracted by binding of the “positive” effector CyPD (which indeed increases the apparent Ca²⁺ affinity of the PTP). Removal of OSCP, or CyPD binding to OSCP, would induce similar conformational effects leading to increased probability of PTP opening, a working hypothesis that awaits experimental verification.

A cartoon with the hypothetical events leading to PTP formation from complex V dimers is presented in Figure ​Figure2.2. In the “coupled” condition the dimers bind Mg²⁺-ADP/ATP, and the concentration of free Ca²⁺ (and Pi, not shown in the Figure) is not high enough for the PT to occur (panel A). Binding of CyPD, which is favored by Pi (Giorgio et al., 2009), would cause a conformational change affecting accessibility of the metal binding sites. This in turn would decrease the matrix Ca²⁺ load necessary to trigger the PT (panels B,D). The transition would essentially depend on replacement of Mg²⁺ with Ca²⁺, which in the absence of CyPD binding would require higher matrix Ca²⁺ loads (panel C). Accessibility of the metal binding site (and probability of occurrence of the PT at a given Ca²⁺ load) would be increased by thiol oxidation and counteracted by thiol reduction. Ion and solute permeation would then occur at the interface between the c rings (panels C,D). The PT can be fully reverted by Ca²⁺ chelation with EGTA (Petronilli et al., 1994), which would restore Mg²⁺ binding and allow the dimer to recover the coupled structure.

Open in a separate window

Figure 2

Hypothetical transition of F_OF₁ ATP synthase dimers to form the PTP. ATP synthase dimers (A) can undergo PTP formation when Ca²⁺ rather than Mg²⁺ is bound, possibly at the catalytic sites, in a reversible process favored by thiol oxidation (C). Binding of CyPD, which is favored by Pi (B) would increase the accessibility of the metal binding sites, allowing PTP formation at lower Ca²⁺ concentrations (as depicted here by a smaller face type) (D). Adenine nucleotides counteract PTP formation in synergy with Mg²⁺. Red arrows denote the hypothetical pathway for solute diffusion between two F_O subunits.

A mystery solved, more mysteries to solve

Conclusions and perspectives

The findings that (1) dimers of the ATP synthase treated with Ca²⁺ generate currents indistinguishable from the MMC–PTP; (2) channel openings are favored by Bz-423 and thiol oxidants; (3) channel openings are inhibited by adenine nucleotides and Mg²⁺; and (4) monomers lack any channel activity, all represent strong indications that the PTP forms from a specific, Ca²⁺-dependent conformation of the dimers. The F_OF₁ ATP synthase readily accommodates key pathophysiological effectors of the PT since Ca²⁺, Mg²⁺, adenine nucleotides, and Pi bind the catalytic core at F₁; and the membrane potential and matrix pH, which are key PTP modulators (Bernardi et al., 2006), are also key regulators of the ATP synthase. On the other hand, several regulators of the PTP (specifically, rotenone and quinones) are not easily accounted for by the dimer hypothesis, and their mechanism of action will need further studies. However, I believe that our findings provide a novel and stimulating working hypothesis that should help address outstanding issues including species-specific features of the PTP of rats (Ricchelli et al., 2005; Zulian et al., 2007), yeast (Jung et al., 1997; Manon et al., 1998; Yamada et al., 2009; Uribe-Carvajal et al., 2011), and Drosophila melanogaster (von Stockum et al., 2011); and the mystery of Artemia franciscana, an anoxia and salt-tolerant brine shrimp whose mitochondria are refractory to PTP opening (Menze et al., 2005, 2010). Together our results suggest a dual function for complex V, ATP synthesis and PTP formation. The key enzyme of life appears therefore to be also the molecular switch that signals the presence of fully depolarized, dysfunctional mitochondria to promote cell death (Rasola and Bernardi, 2011) and/or mitophagy (Youle and Narendra, 2011).

Acknowledgments

References