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

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

Mitochondria have a large Ca2+ problem

In energized mitochondria Ca2+ uptake is an electrophoretic process driven by the Ca2+ electrochemical gradient, :

In respiring, coupled mitochondria the Δψ (negative inside) drives uptake of Ca2+, which is transported with a net charge of 2 [1,2] via an inner membrane channel [3], the mitochondrial Ca2+ uniporter, MCU [4,5]. Ca2+ uptake is charge-compensated by increased H+ pumping by the respiratory chain [1,2], resulting in increased matrix pH that prevents the recovery of Δψ, limiting the further ability to accumulate Ca2+ [6-8]. Uptake of substantial amounts of Ca2+ therefore requires both buffering of matrix pH (to allow regeneration of the Δψ); and buffering of matrix Ca2+ (to prevent the buildup of a Ca2+ concentration gradient) [6-8]. Buffering of matrix pH is achieved by the simultaneous uptake of protons and anions via diffusion of the undissociated acid through the inner membrane (as in the case of acetate), of CO2 (which then regenerates bicarbonate and H+ in the matrix) or through transport proteins (like the H+–Pi symporter) [9]. Buffering of accumulated Ca2+ (and therefore the final [Ca2+] in the matrix) thus depends in part on the cotransported anion and in part on remarkably ill-characterized matrix constituents. If Pi is the prevailing anion, free matrix [Ca2+] becomes invariant with the matrix Ca2+ load [10] and the favors the accumulation of large loads of both Ca2+ and Pi [11], with a predicted Ca2+ equilibrium accumulation of 106 if the Δψ is −180 mV [6]. This is never reached because at resting cytosolic Ca2+ levels the rate of Ca2+ uptake is comparable to that of the efflux pathways, and Ca2+ distribution is governed by a kinetic steady state rather than by thermodynamic equilibrium [6,7]. Thus, in energized mitochondria coupling of Ca2+ uptake with Ca2+ efflux on separate pathways allows regulation of both cytosolic and matrix [Ca2+]. Energy is required both for Ca2+ uptake and for Ca2+ release, owing to the electrophoretic nature of transport on MCU and the 3Na+–1Ca2+ stoichiometry of NCLX [12], which dissipates the Δψ; the requirement for H+ extrusion posed by operation of the Na+–H+ exchanger (NHE) coupled to NCLX; and the fact that Ca2+ efflux on the putative H+–Ca2+ exchanger is favored by the Δψ [13,14], suggesting a H+–Ca2+ stoichiometry higher than 2H+ per Ca2+. A kinetic imbalance is apparent, however, because the Vmax of the MCU is of the order of 1400 nmol Ca2+ mg−1 protein min−1 while the combined Vmax of the efflux pathways is about 20 nmol Ca2+ mg−1 protein min−1. This arrangement exposes mitochondria to the hazards of Ca2+ overload when the rate of Ca2+ uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca2+]. Why is then the rate of Ca2+ efflux so slow?

The rate of Ca2+ uptake via the MCU is a steep function of extramitochondrial [Ca2+] [15]. Increasing rates of Ca2+ efflux would increase extramitochondrial Ca2+, stimulate Ca2+ uptake via MCU and increase overall Ca2+ cycling, resulting in energy dissipation [16]. This can be observed by adding the electroneutral 2H+–Ca2+ ionophore A23187 to respiring mitochondria that have accumulated Ca2+, a condition where Ca2+ is released and all of the respiratory capacity can be diverted into Ca2+ cycling [17]. Thus (and as long as the membrane potential is high) net Ca2+ efflux through stimulation of the efflux pathways would have a high energetic cost. The low Vmax and early saturation of the efflux pathways by matrix Ca2+ are probably designed to pose an upper limit to the energy that can be spent in regulation of matrix and cytosolic [Ca2+] through mitochondrial “Ca2+ cycling”. As mentioned above, however, this situation exposes mitochondria to the constant threat of Ca2+ overload. We have proposed that this event may be prevented by transient openings of the permeability transition pore (PTP), which does mediate mitochondrial depolarization and fast Ca2+ release in vitro and possibly in vivo [18].

Properties of the permeability transition pore in mammals

The PTP is a high-conductance (≈1.3 nS) inner membrane channel [19-21]. In the fully open state its apparent diameter is about 3 nm [22] and its solute exclusion size ≈ 1500 Da. The PTP open-closed transitions are regulated by physiological effectors, and the consequences of pore opening vary dramatically depending on the open time [16]. Here we shall cover only the basic features of PTP modulation, and we refer the Reader to previous reviews for more information on the rise of the PTP from the status of in vitro artifact to that of effector mechanism of cell death regulated by key signaling cascades [23-25].

Matrix modulators of the PTP include Ca2+ through a “permissive” site for opening that can be competitively inhibited by other Me2+ ions like Mg2+, Sr2+ and Mn2+; and Pi, which in most species acts as a powerful PTP inducer through a still undefined mechanism. Pore opening is promoted by an oxidized state of pyridine nucleotides and of critical dithiols at discrete sites, both effects being individually reversed by proper reductants [26]. Pore opening can also cause production of reactive oxygen species, as shown by the occurrence of “superoxide flashes” triggered by transient openings of the PTP in cardiomyocytes [27]. The permeability transition is strictly modulated by matrix pH with an optimum at pH 7.4, while the open probability decreases both below pH 7.4 (through reversible protonation of critical histidyl residues [28,29]) and above pH 7.4 (through an unknown mechanism). Opening of the PTP is inhibited by cyclosporin (Cs) A after binding of the latter to cyclophilin (CyP) D, a matrix peptidyl-prolyl cis-trans isomerase encoded by the Ppif gene that facilitates PTP opening [30-32]; indeed, ablation of CyPD approximately doubles the threshold Ca2+ load required to open the PTP, which becomes identical to that of CsA-treated, strain-matched wild type mitochondria, while no effect of CsA is observed in CyPD-null mitochondria [33-36]. Major membrane effectors are the inside-negative Δψm, which tends to stabilize the PTP in the closed conformation [28]; and electron flux within respiratory chain complex I, with an increased open probability when flux increases [37]. The latter finding led to the discovery that the PT is regulated by quinones [38], possibly through a specific binding site whose occupancy affects the open-closed transitions depending on the bound species [39].

The only primary consequence of PTP opening is mitochondrial depolarization. Unless single channel events are being recorded openings of short duration may not be detected by potentiometric probes. Since opening events are not synchronized for individual mitochondria [40,41] in population studies they may also be missed due to probe redistribution. We refer the reader to a series of specific studies about the occurrence of PTP openings of different durations in mitochondria in situ, and their consequences on cell viability [40-44]. For openings of longer duration depolarization can be easily measured both in isolated mitochondria and intact cells. As long as the pore is open collapse of the prevents ATP synthesis, and ATP hydrolysis by the mitochondrial ATPase worsens ATP depletion, which together with altered Ca2+ homeostasis is a key factor in various paradigms of cell death [45]. Persistent PTP opening is also followed by loss of matrix pyridine nucleotides with respiratory inhibition [46], by equilibration of ion gradients, and by diffusion of solutes with molecular masses lower than about 1500 Da and possible occurrence of swelling, cristae unfolding and outer membrane rupture. This, however, is not an inevitable consequence of PTP opening. Cristae remodeling due to PTP opening can also occur in the absence of outer membrane rupture [47] with mobilization of the large pool of cytochrome c usually compartmentalized in the intracristal spaces [48] allowing increased release of cytochrome c through BAX/BAK channels in an otherwise intact outer membrane [47]. Furthermore, osmotic swelling requires the existence of a colloidosmotic gradient between the matrix and the intermembrane space that may not exist (or may not be large enough) to cause outer membrane rupture in situ.

The possible role of the PTP in physiological Ca2+ homeostasis has not been studied thoroughly, in part because the nature of the PTP has remained elusive [23] and therefore modulation of the “channel” itself by genetic methods has not been possible; in part because true PTP blockers have not been developed. Great hopes were raised by the discovery that the PTP is inhibited by CsA [49-51], but it is now clear that CyPD is a key modulator but not an obligatory component of the PTP. It should therefore be borne in mind that Ppif−/− (CyPD-null) mice are not PTP-null mice, as a permeability transition can still occur, e.g. after an increased Ca2+ load [33-36]. This consideration has dramatic implications for the interpretation of results obtained with CsA; similar to the absence of CyPD, CsA can desensitize but not block the PTP, and therefore lack of sensitivity to CsA does not necessarily mean that the PTP is not involved in the event under study. Little attention has also been paid to the fact that expression of CyPD can be modulated (e.g. by muscle denervation [52]), and that only CyPD-expressing mitochondria are expected to respond to CsA. A further element of complexity is that CsA also affects mitochondria in situ by preventing calcineurin-dependent dephosphorylation of the pro-fission protein DRP1, which is essential for its translocation to mitochondria [53]. The resulting inhibition of mitochondrial fission by CsA may thus be a cytoprotective event that is independent of inhibition of CyPD and desensitization of the PTP [53], a finding that should induce some caution in evaluating in situ and in vivo studies based only on CsA rather than on genetic ablation of CyPD or on the use of CyP inhibitors devoid of effects on calcineurin [54-56].

The mitochondrial permeability transition pore as a Ca2+ release channel: is lack of selectivity a problem or an advantage?

If a Ca2+ concentration gradient exists between the matrix and the external medium (or the cytosol) PTP opening leads to Ca2+ release. In 1996 we have proposed that the PTP may serve as a mitochondrial Ca2+ release channel [18], and a specific point that needs to be discussed is whether the lack of selectivity is a real problem, or rather an essential feature that allows fast and effective release of matrix Ca2+. Ca2+ efflux down its concentration gradient via a Ca2+-selective channel would be opposed by the buildup of a Ca2+ diffusion potential [57]. According to the Nernst equation, the magnitude of the Ca2+ diffusion potential when no charge-compensating species are present is −30 mV per decade of the matrix/cytosol Ca2+ concentration ratio. This diffusion potential is reduced to less negative values (with corresponding increase of the Ca2+ efflux rate) by any charge-compensating current (influx of positive charges, efflux of negative charges, or both). Since the inner membrane has a very low permeability to charged species, the rate of Ca2+ efflux would be extremely slow and essentially limited by the H+ permeability. In other words, to obtain a significant rate of Ca2+ efflux via a Ca2+-selective channel the inner membrane permeability should be increased as well. An unselective pore of large size like the PTP confers the advantage of providing charge compensation within the channel itself, thus allowing maximal Ca2+ flux (i.e. Ca2+ release would occur at zero potential). This would make Ca2+ release possible even for vanishingly small [Ca2+] gradients, regulation being achieved through modulation of the pore open time. It should be noted that no K+ and Na+ concentration gradients exist across the inner membrane because the slow electrophoretic uptake of K+ and Na+ is compensated by the K+–H+ exchanger and the NHE, respectively. Thus, PTP opening can lead to selective Ca2+ release without perturbation of K+ and Na+ homeostasis, and no evolutionary pressure may have existed for the development of cation selectivity. In this respect the PTP is strikingly similar to the Ca2+ release channel of the sarcoplasmic reticulum, which operates as a Ca2+-selective channel despite its large size (>38 Å), high conductance for monovalent cations (≈1 nS at saturating K+), permeability to solutes like glucose and low permeability ratio when both K+ and Ca2+ are present (PCa/PK ≈ 6) [58].

A Ca2+ release channel in Drosophila mitochondria: the missing link between the PTP of yeast and mammals?

We have recently argued that, in spite of clear differences between species, the PTP has been conserved in evolution from yeast to mammals [59]. Yeast mitochondria do possess a permeability pathway that resembles the mammalian PTP, also called the Yeast Mitochondrial Unselective Channel (YMUC) [60,61]. Based on the features of the PTP of yeast and mammals as of 1998, Manon et al. concluded that regulation of YMUC is too different from that of the mammalian PTP for yeast to be a good model of the latter [60]; in the light of new results we believe that the differences are not as fundamental as suspected earlier, and we refer the reader to a recent review specifically devoted to this aspect [59]. Other than in mammals and yeast, occurrence of a permeability transition has been established in plants, amphibians and fish including zebrafish (Danio rerio) [62] (see [59] for references). A mitochondrial Ca2+ release channel (mCRC) that we recently identified in cells from Drosophila melanogaster [63] deserves a special mention, as it may represent an evolutionary variant of the PTP displaying remarkable selectivity toward Ca2+ and H+.

Mitochondria were studied in digitonin-permeabilized S2R+ cells [63], which are derived from Drosophila late embryonic stages and represent a variety of tissue precursors [64]. Of interest, mitochondria of S2R+ cells possess all the classical Ca2+ transport pathways found in mammalian mitochondria, i.e. (i) the RR-sensitive Ca2+ uniporter (which is consistent with the existence of orthologs of MCU [4,5] and of MICU1 [65] in the Drosophila genome); (ii) the Na+–Ca2+ antiporter recently identified as NCLX [12], whose ortholog also exists in Drosophila; (iii) the RR-insensitive putative H+–Ca2+ antiporter mediating Ca2+ release at high membrane potential; (iv) a tetracaine-sensitive, RR-insensitive Ca2+ release pathway that opens in response to matrix Ca2+ loading or to depolarization, and mediates Ca2+ release [63]. The properties of the Drosophila mCRC appear to be intermediate between those of the PTP of mammals and yeast. Like the mammalian PTP, the Drosophila Ca2+ release pathway is inhibited by tetracaine [66] and opens in response to matrix Ca2+ loading, inner membrane depolarization, thiol oxidation and treatment with relatively high concentrations of NEM [63]. Like the yeast PTP (and at striking variance from the mammalian pore) the Drosophila mCRC is inhibited rather than stimulated by Pi; and is insensitive to ADP, quinones and to CsA [63], a finding that matches the absence of a mitochondrial CyP in Drosophila. Together with the inhibitory effect of Pi, the most striking difference between the Drosophila mCRC and the PTP is the selectivity for the transported species. At the onset of Ca2+-dependent Ca2+ release Drosophila mitochondria undergo depolarization, suggesting that the putative channel is also permeable to H+; yet no matrix swelling is observed even in KCl-based medium, indicating that the channel is not permeable to K+ (and to Cl−) in spite of the fact that the hydrated radius of Ca2+ is larger than that of K+ [63]. Lack of swelling was confirmed by lack of cytochrome c release and by ultrastructural analysis, and was not due to peculiar structural features of Drosophila mitochondria because matrix swelling and cytochrome c release readily followed the addition of the pore-forming peptide alamethicin [63].

The key features of the yeast and mammalian PTP, and of the Drosophila mCRC are summarized in Table 1. The most remarkable differences are the permeability to solutes, the selectivity for Ca2+ and the effects of Pi. Since nearly all yeast strains lack an MCU, the Ca2+-dependence of the yeast PTP has not been easy to assess although it was known that the yeast PT is favored by added Ca2+ [67]. Recent work using the Ca2+ ionophore ETH129, which mediates electrophoretic Ca2+ transport, has demonstrated that the PTP is favored by Ca2+ uptake in mitochondria from Saccharomyces cerevisiae [68], and based on this finding we suggest that matrix Ca2+ may be required for onset of the PT in yeast as well (Table 1). Lack of inhibition of Drosophila mCRC by CsA can be easily explained by the lack of a mitochondrial CyP. On the other hand, since a matrix CyP is found in yeast and its enzymatic activity is inhibited by CsA [69] we suspect that the CyP ability to interact with the PTP occurred later in evolution.

Table 1

Properties of mammalian and yeast PTP, and of the Drosophila mCRC.

	Mammals	Yeast	Drosophila
Permeability to solutes up to ≈1500 Da	Yes	Yes	No
Selective Ca2+ release	No	No	Yes
Matrix Ca2+	Required	May be required	Required
Matrix Pi	Inducer	Inhibitor	Inhibitor
mt Cyp	Yes	Yes	No
CsA	Inhibitor	No effect	No effect
Tetracaine	Inhibitor	Not tested	Inhibitor
Redox sites	Yes	Yes	Yes

The PTP as a mitochondrial Ca2+ release channel: only a hypothesis?

In 1992, Altschuld et al. demonstrated that CsA significantly increases net Ca2+ uptake and decreases Ca2+ efflux in isolated cardiomyocytes, as measured by radiolabeled 45Ca2+, without having any impact on cell morphology or viability [70]. The effect of CsA was concentration-dependent and specific to mitochondria, as ATP-dependent Ca2+ uptake by the sarcoplasmatic reticulum was not affected. The selectivity of the effects for mitochondrial Ca2+ and the very short incubation time with CsA (15 min) suggest that the PTP was being affected, and these data may represent the first piece of evidence that the pore contributes to Ca2+ cycling in mitochondria of living cardiomyocytes, and that reversible pore opening may be a physiological process in heart cells [70]. On the other hand, Eriksson et al. found that fluxes of Ca2+, Mg2+ and adenine nucleotides in perfused rat livers following hormonal stimulation were unaffected by previous administration of CsA, even if the PTP was demonstrably desensitized by CsA in mitochondria isolated from the same CsA-perfused livers [71]. The Authors concluded that regulation of mitochondrial ion and metabolite homeostasis is independent of the PTP [71], yet as discussed above a negative result is not as informative.

Two recent publications based on Ppif−/− cells and mice do provide clear support for a role of the PTP in Ca2+ homeostasis [72,73]. Adult cortical neurons from wild type and Ppif−/− mice were treated with either ATP (to activate P2Y purinergic receptors) or with depolarizing concentrations of KCl (to open plasma membrane voltage-dependent Ca2+ channels), both stimuli causing a robust increase of both cytosolic and mitochondrial [Ca2+] that was indistinguishable in neurons of the two genotypes [73]. Application of the two stimuli together, however, resulted in much higher levels of mitochondrial [Ca2+] in the Ppif−/− neurons, suggesting that the threshold for PTP activation had been reached in the wild type but not in the CyPD-null mitochondria in situ. Thus, it appears that the regulatory role of CyPD (and of PTP opening) becomes crucial only for relatively large mitochondrial Ca2+ loads exceeding the capacity of NCLX and of the Na+-insensitive release pathway [73]. In other words, the PTP could be silent unless high Ca2+ loads saturate the NCLX and the H+–Ca2+ exchanger, allowing matrix [Ca2+] to rise enough to trigger pore opening. It should be noted that the transient stimulation of these Ca2+-mobilizing pathways did not induce cell death either in wild type or in CyPD-null neurons, suggesting that the mitochondrial PTP-activating response was part of a physiological process that is consistent with occurrence of reversible PTP opening [42].

In adult mice ablation of CyPD increases resistance to acute ischemia-reperfusion injury both in the heart and brain [33,35,36,74], while in neonatal mice, where the membrane-permeabilizing effects of BAX predominate, Ppif−/− mice were instead remarkably sensitized suggesting age-related changes in the mitochondrial response to injury [74]. An age-related phenotype was also discovered in the hearts of Ppif−/− mice, which displayed an intriguing decrease of maximum contractile reserve matched by increased shortening and relaxation times with longer decay of cytosolic Ca2+ transients [72]. Ppif−/− mice were also unable to compensate for the increase in afterload caused by transaortic constriction, displaying a larger reduction in fractional shortening, decompensation, ventricular dilation, fibrosis and congestive heart failure; all consequences of CyPD ablation were cured by heart-selective reexpression of CyPD, strongly indicating that the maladaptive phenotype of Ppif−/− mice depends on a primary disturbance of myocyte mitochondria rather than on an underlying systemic response [72]. Metabolic in vivo analysis demonstrated a significant increase in the glucose to palmitate ratio, suggesting a metabolic shift from fatty acid oxidation to glycolysis associated with increased activity of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Direct measurement of total mitochondrial Ca2+ content of Ppif−/− hearts showed a 2.6-fold increase, which was matched by greater mitochondrial Ca2+ transients in myocytes treated with CsA. Very importantly, under continuous pacing PTP desensitization with CsA decreased the rise time in Ca2+ accumulation and prolonged the recovery time after pacing, findings that are entirely consistent with the PTP acting as a Ca2+ release channel to prevent Ca2+ overload [72].

Summary and conclusions

The hypothesis that transient opening of the PTP may serve the physiological function of regulating matrix Ca2+ by preventing Ca2+ overload appears theoretically justified, and supported by a limited but extremely solid set of experimental results. Confusion may have arisen from the interpretation of results based on the use of CsA as well as of Ppif−/− mice, which have too often been erroneously considered null for the PTP as well. Much more work is obviously needed, and we hope that this short review will help rekindle interest and experimental testing of this subject.