Inhibition of the Mitochondrial Permeability Transition by Creatine Kinase Substrates

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by β-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by ␤-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
Most vertebrate cell types express cytosolic as well as mitochondrial isoforms of the enzyme creatine kinase (CK). 1 CK catalyzes the reversible transphosphorylation of phosphocreatine (PCr) to ATP. The findings of distinct subcellular localizations of creatine kinases have led to the formulation of the PCr circuit concept, proposing that sites of energy production (mitochondria and glycolysis) are tightly linked via Cr/PCr shuttling to sites of energy consumption (various cellular ATPases) (1)(2)(3)(4)(5)(6). ATP generated by oxidative phosphorylation in mitochondria reacts with creatine (Cr) to produce PCr, a reaction mediated by mitochondrial CK (mtCK) located in the intermembrane and intercristae space. Phosphocreatine then diffuses to the cytosol to locally regenerate ATP via cytosolic CK from ADP. Cr produced during this reaction is shuttled back to the mitochondria for recharging it to PCr. This energy shuttling system is particularly efficient in tissues with a very high and fluctuating energy demand like skeletal and cardiac muscle, as well as in brain and neural tissues (7). Thus, in addition to the generally accepted temporal energy buffering function, PCr also provides a means to spatially buffer energy reserves (3). This holds especially true for highly polar cells like spermatozoa where the diffusion of ADP is the limiting factor (8).
Studies with cultured rat hippocampal neurons have shown recently that creatine protects against glutamate and ␤-amyloid toxicity (9), as well as against energetic insults in striatal neurons (10). Similarly, creatine and in some cases the related analog cyclocreatine (CyCr) exert protective effects in several animal models of neurodegenerative diseases, like Huntington's disease (11), amyotrophic lateral sclerosis (12), and a form of Parkinsonism (13). Likewise, Cr pretreatment of cultured myotubes from dystrophic mdx mice enhanced myotube formation and survival (14). Recent data indicate that Cr reduces muscle necrosis and protects mitochondrial function in vivo in mdx mice (15). Treatment of patients with Cr or Cr analogs has, therefore, been proposed as a possible adjuvant therapy for such diseases (16). The protection observed with Cr in these cell and animal models may be explained partially by its function as a cellular energy buffer and transport system via the PCr shuttle as outlined above. An additional potential mechanism of Cr protection may be linked to direct effects on mitochondrial permeability transition (MPT) (17), which has been suggested to be a causative event in different in vivo and in vitro models of cell death (18 -24).
In an attempt to define the role of mitochondrial creatine kinase on MPT, we have shown in an earlier study that isolated liver mitochondria from transgenic mice containing mtCK did not respond by MPT pore opening upon treatment with Ca 2ϩ plus atractyloside if Cr or CyCr were present in the medium (17). In the absence of Cr and CyCr, however, MPT pore opening could be fully induced by Ca 2ϩ plus atractyloside. On the other hand, in liver mitochondria from control mice without mtCK, pore opening induced by Ca 2ϩ plus atractyloside was independent on the presence or absence of Cr or CyCr. In the present study we investigated these effects in more detail, asking the question of whether inhibition of MPT pore opening by CK substrates depends on mtCK activity and whether the tight functional coupling between the CK reaction and oxidative phosphorylation, demonstrated recently to take place in situ (25), would lead to cycling of mitochondrial adenine nucleotides, thus resulting in net production of phosphorylated CK substrates. Our results indicate that substrate channeling between mtCK and adenine nucleotide translocase (ANT) takes place in a tight functionally coupled microcompartment that seems absolutely required for protection of MPT pore opening by creatine.

EXPERIMENTAL PROCEDURES
Source and Preparation of Mitochondria-Transgenic mice expressing the ubiquitous mitochondrial creatine kinase isoform in their liver mitochondria (transgenic liver-mtCK mice) were kindly provided by Dr. Alan P. Koretsky (National Institutes of Health, Bethesda). Mice were killed, and the liver was quickly removed and placed in ice-cold isolation buffer for mitochondria (10 mM Tris-HCl, pH 7.4, 250 mM sucrose) supplemented with 1 mM EDTA and 0.1% bovine serum albumin. Livers were homogenized, and the homogenate was centrifuged at 700 ϫ g for 10 min. The supernatant was filtered through two layers of nylon gauze and centrifuged at 7000 ϫ g for 10 min. The mitochondrial pellet was washed twice with isolation buffer (without EDTA and bovine serum albumin), centrifuged, and kept on ice.
Respiration and Swelling Measurements-Mitochondrial oxygen consumption was measured with a Cyclobios oxygraph (Anton Paar, Innsbruck, Austria) at 25°C. The standard medium (2 ml) consisted of 10 mM Tris/Mops, pH 7.4, 250 mM sucrose, 10 mM phosphate/Tris, 2 mM MgCl 2 , 0.5 mM EGTA/Tris, 2 M rotenone, and 50 M Ap 5 A. State 4 respiration was stimulated by addition of 5 mM succinate/Tris. Creatine kinase substrates (Cr, CyCr, and GPA) were present at 10 mM concentration, and state 3 respiration was induced by adding 1 mM ATP. Mitochondrial protein concentration in all assays was 0.5 mg/ml. Deviations from these conditions are specified in the figure legends.
Mitochondrial MPT pore opening was measured by the convenient swelling assay and carried out in a UV4 UV/Vis spectrometer (Unicam) connected to a computer. Swelling curves were recorded at 540 nm, and data points were acquired every 1/8 s using Vision © software (version 3.10, Unicam Ltd.). Cuvettes containing the mitochondrial suspension were thermostated to 25°C. Incubation conditions and induction of MPT are indicated in the legends to the figures.
Measurement of PCr Production by Quantitative Thin Layer Chromatography-Mitochondria were incubated under different conditions (see legend to Fig. 4) in the presence of 32 P i . Reactions were stopped after defined time intervals by addition of 1% (final concentration) SDS and centrifuged, and the supernatants were applied to Silica Gel 60 thin layer plates (Merck). Running solvent was a mixture of isopropyl alcohol, ethanol, and 25% ammonia (6:1:3, by volume). Thin layer plates were air-dried, exposed to a Kodak storage phosphorscreen SO230, and analyzed with a PhosphorImager (Storm 820, Amersham Biosciences). The position of PCr in thin layer chromatograms was identified in control experiments using recombinant brain-type BB-CK incubated in the presence of radioactive [ 14 C]creatine and cold ATP.
Binding of Exogenously Added CK to Mitochondria-Mouse liver mitochondria (0.5 mg/ml) were incubated in standard medium at pH 7.4 with 5 mM glutamate and 2.5 mM malate and without rotenone. Specified amounts of recombinant brain-type BB-CK or ubiquitous human mtCK (prepared as described elsewhere (26,27)) were added. After a 5-min incubation, an aliquot of the suspension was removed (to measure total CK activity). The rest was centrifuged (5 min at 7000 ϫ g) to separate mitochondria. CK activity was measured separately in the supernatants and mitochondrial pellets by a coupled enzymatic assay (28).
Other Methods-Protein concentrations were determined by Bradford assay (Bio-Rad) using bovine serum albumin as a standard. Mitochondrial adenine nucleotide content was measured by reversed phase chromatography of acid extracts according to Kay et al. (25).

RESULTS
To correlate the effects of the different creatine kinase substrates on MPT pore opening with their ability to stimulate oxidative phosphorylation, we first measured stimulation of state 3 respiration by ATP and CK substrates in mitochondria oxidizing succinate. The data of these experiments are summarized in Table I. In mtCK containing mitochondria from transgenic mice, substantial stimulation (about 3-fold over state 4) was observed only with Cr and CyCr (both 10 mM), but not with GPA, in the presence of externally added ATP, due to endogenous production of ADP by mitochondrial CK. The absence of a detectable stimulation with 10 mM GPA agrees with the inability of mitochondrial CK to phosphorylate this creatine analog (29,30). No Cr-or CyCr-stimulated respiration was seen with mitochondria from control mice, which do not express mtCK in liver (17). These data confirm earlier findings with the same substrates given to heart mitochondria (30). In general, with respect to creatine-stimulated respiration, mitochondria from transgenic liver-mtCK mice are behaving comparably to mtCK containing heart mitochondria (see also Ref. 17).
Next, we measured, in transgenic mtCK-liver mitochondria, the opening of the permeability transition pore induced by Ca 2ϩ in the presence of CK substrates, but in the complete absence of external adenine nucleotides, and under different of conditions. To avoid any effects caused by adenylate kinase activity, the specific adenylate kinase inhibitor Ap 5 A was included in the medium in all experiments. In the experiments shown in Fig. 1A, mitochondria were energized with glutamate and malate in the absence of rotenone. Due to the presence of 2 mM Mg 2ϩ (but no EGTA) in the medium, a rather high (120 M) Ca 2ϩ pulse had to be administered to overcome MPT inhibition by magnesium. As shown in Fig. 1A, Cr and CyCr effectively inhibited pore opening within the time frame of the experiment. In the absence of CK substrates or with GPA present, MPT pore opening occurred in a significant fraction of the mitochondrial population. These effects of CK substrates were independent, at least qualitatively, on how the MPT was triggered and of the respiratory substrates used as documented in Fig. 1, B and C. In the experiments shown in Fig. 1B, mitochondria were again energized with complex I substrates (glutamate and malate), but exposed to only 40 M Ca 2ϩ (which did not open the MPT per se). Subsequent depolarization with 0.2 M FCCP led to rapid swelling of the mitochondria, both in the absence and presence of CK substrates. Swelling was again sensitive to CsA, as well as to 50 M ubiquinone 0, a novel and general MPT inhibitor (not shown) (31), indicating opening of the MPT pore. Remarkably, however, Cr and CyCr exerted MPT protection in a significant subpopulation of mitochondria even in the absence of external adenine nucleotides and under these very strongly pore-promoting conditions (absence of EGTA, high phosphate, depolarization of mitochondria by FCCP, and presence of complex I substrates). Again, GPA did not protect mitochondria from MPT pore opening. Fig. 1C shows the same set of experiments but with succinate-energized mitochondria in the presence of rotenone. Here, the same qualitative conclusions can be drawn as from the data presented in Fig. 1, A and B. In accordance with the finding of Fontaine et al. (32), conditions are more restrictive for MPT pore opening with complex II-linked substrates due to decreased electron flux through complex I, a general property of the MPT. With succinate as the electron donor, Cr and CyCr, but not GPA, fully protected from MPT pore opening. Next we analyzed MPT protection by creatine at different calcium concentrations. In the experiments displayed in Fig.  1D, the Mg 2ϩ concentration was reduced to 0.5 mM to compare better the effect of Cr as a function of the calcium load. Under these conditions, 30 M Ca 2ϩ did not open the MPT pore if Cr was present. Pore opening was, however, observed at higher Ca 2ϩ concentrations (90 and 150 M, solid curves in Fig. 1D). Nevertheless, Cr still inhibited significant fractions of mitochondria when compared with conditions without Cr but equal Ca 2ϩ concentrations (dashed curves in Fig. 1D).
Based on these observations and the data presented in Table  I, we suggested that the protective effect on MPT pore opening seen with Cr and CyCr as compared with GPA could be related to the kinetic properties of these substrates, i.e. their rate of phosphorylation by mtCK. In contrast to GPA, Cr and CyCr are rapidly converted by mtCK via ATP to their respective phosphorylated compounds, PCr and CyPCr (30). If substrate phos-phorylation by mtCK (with internally available ATP) were responsible for the observed effects, protection of MPT by Cr and CyCr should have been abolished under conditions where CK is inactive as an enzyme. As there is no absolutely specific CK inhibitor available, we omitted Mg 2ϩ instead, which is an essential cofactor for the CK reaction (33), from the medium. With no Mg 2ϩ present, creatine-stimulated respiration with ATP was almost completely absent as shown in Fig. 2A (compare slopes at arrow in trace a with that of trace b). Importantly, in the absence of exogenous Mg 2ϩ , state 3 respiration was still observed after addition of ADP (disodium salt, Fig. 2A, trace a) indicating that sufficient matrix Mg 2ϩ is available for phosphorylation of ADP by F 0 F 1 -ATP synthase, but this Mg 2ϩ is not accessible to mtCK located in the intermembrane space. As shown in Fig. 2B, in the absence of Mg 2ϩ , mitochondrial swelling induced by 20 M Ca 2ϩ and 0.2 M FCCP occurred to the same extent regardless of whether CK substrates were present or not, in contrast to what was observed in the presence of Mg 2ϩ (see the corresponding traces in Fig. 1B). MPT pore opening, however, was still fully blocked by 1 M CsA, even in the absence of Mg 2ϩ .
In a further set of experiments, we analyzed the effect of Mg 2ϩ and Cr on MPT in more detail by varying the concentrations of these substrates and measuring mtCK activity under identical conditions. Representative swelling measurements at two different Mg 2ϩ concentrations with (solid curves) and without (dashed curves) Cr are displayed in Fig. 3A. From such curves we determined the absorption difference (⌬A 540 ) before and 4 min after triggering the MPT with 120 M Ca 2ϩ (same conditions as in the experiments of Fig. 1A). The ⌬A 540 values were used to calculate the fraction of swollen mitochondria with reference to the ⌬A 540 measured with 5 M of the channelforming peptide alamethicin, which resulted in (not MPTcaused) swelling of 100% of the mitochondria (not shown). As shown in Fig. 3C, the fraction of swollen mitochondria, i.e. the fraction having undergone a permeability transition, decreased at increasing Mg 2ϩ concentrations, also in the absence of Cr (open circles in Fig. 3C). This was to be expected, as the MPT is regulated by an external inhibitory Me 2ϩ -binding site (34). However, in the presence of 10 mM Cr, an additional protection starting at around 0.5 mM Mg 2ϩ is observed (closed circles in Fig. 3C). At this Mg 2ϩ concentration, mtCK activity, as measured in the pH-stat in the forward reaction with ATP plus Cr,  arrows in traces b and a, respectively). B, swelling measurements. Incubation conditions were as specified in the legend to Fig. 1A, except that MgCl 2 was omitted. CK substrates (Cr or CyCr) were present at 10 mM concentration. In the uppermost trace, the medium was supplemented with 1 M CsA. Reactions were started by addition of 0.5 mg of mitochondria (M) to 1 ml of medium followed by Ca 2ϩ and FCCP as indicated.
was at about 70% of the maximum value (open triangles in Fig.  3C). Therefore, MPT protection by Cr shows up at relatively high Mg 2ϩ concentrations only (Ͼ0.5 mM) with corresponding high mtCK activities. A similar analysis carried out by variation of the Cr concentration at constant [Mg 2ϩ ] (2 mM) revealed that MPT protection by Cr is clearly a function of mtCK activity (Fig. 3, B and D).
These data are in strong support of the idea that the rate of phosphorylation of CK substrates by active mtCK is related to their effect on MPT. Phosphorylation of these CK substrates occurs in microcompartments formed by mtCK and ANT (see below) (35). Because we did not add external adenine nucleotides, phosphorylation must occur via internally available ATP inside mitochondria, suggesting continuous cycling of internal ADP and ATP between matrix and intermembrane space mediated by ANT, if Cr or CyCr are present. As a consequence, if Cr is present, we should expect a net production of PCr even in the absence of exogenously added adenine nucleotides. This is indeed the case as shown in Fig. 4A. Mitochondria were incubated in the presence of 32 P i , Mg 2ϩ , and 10 mM creatine. Only with energized mitochondria and fully active mtCK, as well as with a working oxidative phosphorylation system, we could observe a generation of PCr (Fig. 4, lane a). These are exactly the conditions used in the swelling experiments where MPT protection by Cr was observed (Fig. 1). With de-energized mitochondria (Fig. 4, no substrate, lane b), blocked ANT (20 M atractyloside, lane d), or blocked F 0 F 1 -ATP synthase (1 M oligomycin, lane e), no PCr was produced. Note in lane c (absence of Mg 2ϩ ), some PCr is still generated due to residual Mg 2ϩ (probably bound to mtCK). Fig. 4B shows a time course experiment of net PCr production by energized mitochondria in the presence of external Cr plus Mg 2ϩ , but no adenine nucleotides. The measured adenine nucleotide content is 0.11 Ϯ 0.01 nmol per 25 g of isolated mitochondria from transgenic liver-mtCK mice. After a 40-min incubation of succinate-energized mitochondria, 2.85 nmol of PCr were produced by 25 g of mitochondria (Fig. 4B). Recalling that one ATP is consumed for every molecule of PCr produced, we can estimate that this is over 25 times more than the measured adenine nucleotide content. This quantitative consideration is a strong argument for internal nucleotide cycling.
Mitochondrial CK forms a microcompartment with ANT at the contact sites (together with outer membrane porin), as well as along the cristae (with ANT only (36,37)). This compartmentation allows efficient transphosphorylation of ATP to PCr and export of the latter to the cytosol at peripheral contact sites. The reaction product ADP is fed back via ANT to the matrix for rephosphorylation resulting in an overall lowering of the apparent K m values of ADP for oxidative phosphorylation (38). It is conceivable that microcompartmentation of mtCK and ANT is also responsible for the observed MPT inhibition by Cr and CyCr (48). To test this idea, we measured MPT pore opening in liver mitochondria from control mice lacking mtCK but with exogenously added recombinant human brain-type dimeric CK (BB-CK). Under the conditions used, especially at pH 7.4, this cytosolic isoform does not bind to mitochondrial outer membranes (Fig. 5A). The amount of BB-CK enzyme activity added in these experiments was equivalent to that found (based on mtCK activity) within the mitochondria of transgenic liver-mtCK mice. Under these conditions, no protection of MPT pore opening of control mitochondria with externally added CK by any of the CK substrates was seen, even in the presence of Mg 2ϩ (Fig. 5B, shown only for Cr). By contrast, with the same amount of ubiquitous mtCK (the isoform expressed in the liver mitochondria of the transgenic mice), significant binding of the enzyme to the surface of mitochondria was observed (Fig. 5A). However, even with mtCK bound to the outside of mitochondria, no noticeable MPT protection by Cr was observed (Fig. 5C) as was also the case for BB-CK (Fig. 5B). Even increasing the total amount of mtCK by 10-fold in the medium did not bring about detectable MPT protection by Cr, although absolute binding of mtCK to the mitochondrial sur- face increased by more than 3-fold under these conditions (Fig.  5A). Thus, addition of CK externally or even mtCK bound to the outer membrane was not able to confer significant protection against MPT. DISCUSSION The present study was carried out to provide a mechanistic basis for the observation that certain substrates of creatine kinase efficiently inhibit the MPT (17). Because opening of the MPT pore appears to be causally related to cell death in several models (18 -24), these studies may provide the basis for novel cytoprotective drugs. We have measured MPT pore opening in isolated mitochondria by the convenient swelling assay in sucrose-based medium and under different conditions, and we compared the response of transgenic mitochondria containing active or inactive CK with that of control mitochondria to which CK was added externally. These experiments revealed a major difference in MPT behavior. With liver mitochondria from transgenic mice, expressing mtCK in these organelles (39), but not with liver mitochondria from wild-type mice, we have observed MPT inhibition (based on measurements of uncoupled respiration) in the presence of Cr or CyCr in an earlier study (17). Here, by using mitochondrial swelling assays, we show that MPT protection by these CK substrates critically depends on two key factors.
The most striking new findings from the present study are that Cr protection of MPT requires magnesium, an essential cofactor for the CK reaction, and active mtCK at its proper location in the IMS. These observations together with the finding of net PCr production by isolated mitochondria, even in the absence of exogenous adenine nucleotides, suggest that endogenous adenine nucleotides are permanently cycling via ANT between matrix and IMS, if CK substrates are present that can efficiently be phosphorylated. This is the case for Cr and CyCr but not for GPA.
The consequences for MPT modulation by CK substrates could then be explained by the influence of nucleotide binding to ANT and conformational changes of this carrier (40,41). To visualize this (see Fig. 6), during adenine nucleotide exchange, the common transport site for ADP and ATP on the ANT faces alternatively the matrix (m) and cytosolic (c) side (42,43). Accordingly, the ANT changes its conformation between mand c-state, if conditions allow adenine nucleotide transport. In the absence of a protonmotive force, the matrix ATP/ADP ratio of isolated mitochondria is low (40,44), and it is entirely conceivable that adenine nucleotides are enzyme-bound (e.g. to the ATP synthase). Certain (unknown) fractions of transport units (shown as ANT dimers in Fig. 6, see also Ref 45) are locked either into the m-or c-state (Fig. 6A). Upon energization, the matrix ATP/ADP ratio rises, and ATP will be liberated and transported to the IMS. There, ATP either gets diluted in the medium or is trapped by mtCK (Fig. 6B). As a consequence, more transport units would now be in the c-state than before energization. Under these conditions, MPT pore opening should be favored, as seen with no CK substrate present. This situation is likely to be similar to the effect of the strong ANT inhibitor, atractyloside, known to stabilize the c-conformation and being an inducer of the MPT (46). If, however, Cr (or CyCr) is present, the trapped ATP will be transphosphorylated and the generated ADP transported to the matrix for rephosphorylation (Fig. 6C). As this process proceeds, the time-averaged fraction of transport units occupied with adenine nucleotides and being in the m-state is expected to be higher, and therefore, conditions for MPT pore opening are less favorable. In the presence of GPA, the ATP delivered to the active site of mtCK is not used up because GPA is not phosphorylated by mtCK (29). Consequently, the ANT is largely locked into the c-conformation, again favoring MPT pore opening. At present, it is still unclear how ANT conformation would affect the MPT. An indirect effect, e.g. via the surface potential, has been proposed (41,47). Note the emphasis on microcompartmentation and functional coupling of mtCK and ANT as an essential part of the model shown in Fig. 6. This is corroborated by the fact that similar amounts of either cytosolic BB-CK or mtCK added externally to the outside of control mitochondria, as is present as mtCK in transgenic mitochondria, did not result in any significant Cr protection of MPT.
We have clearly demonstrated that in the presence of Cr, by measuring net production of PCr, oxidative phosphorylation proceeds even without adding external adenine nucleotides, although at a slow rate only. Nevertheless, besides the effect on the conformational state of the ANT, an additional contribution to MPT protection by Cr could be caused by variation of the matrix ATP/ADP ratio in favor of ADP (25), which is a strong MPT inhibitor (40,44). On the other hand, the accumulating PCr is not believed to exert MPT inhibition (e.g. via the membrane potential) as we have shown earlier (17).
As mtCK functionally interacts with ANT at mitochondrial contact sites, as well as along the cristae membranes (36), a second possibility is that the two proteins may interact and that modulation by CK substrates may affect pore formation by the ANT. The known property of ANT to form an unspecific pore showing some of the characteristics of the MPT pore (48 -50) in in vitro reconstituted systems has been taken by several authors as evidence that the ANT represents the central element of an MPT pore complex (51-59). However, it should be considered that pore formation is not unique to ANT but has also been described for other members of the mitochon- drial carrier family (60,61). Furthermore, mitochondria from ANT-deficient yeast still exhibit a mitochondrial multiconductance channel which is believed to be the electrophysiological counterpart of the MPT (62). A recent study by Linder et al. (63), showing that arginine modification has pronounced influences on MPT pore opening that are not modulated by the ANT ligands atractyloside and bongkrekic acid, further questions a direct involvement of the ANT in pore formation. Finally, the structural interactions between mtCK and ANT that were postulated in models of mitochondrial contact sites (64) still await experimental proof.
Creatine has been shown to exert strong protective effects against glutamate and ␤-amyloid toxicity and energetic insults in neuronal cell cultures (9,10), as well as in several animal models of neurodegenerative diseases (11)(12)(13). It is interesting to note that in the study of Brustovetsky et al. (10), Cr did not prevent MPT in isolated brain mitochondria, despite protection from energetic insults in cultured striatal neurons. This could be either due to the low magnesium concentration (0.5 mM) used by these authors in their assays or to the only weak coupling of the CK reaction to oxidative phosphorylation in these particular preparations. For the latter, however, no data were provided. In any case, both interpretations are fully in line with the model concerning the effect of Cr on MPT as proposed above. Thus, the beneficial effects of Cr seen in animal disease models can be attributed to at least two mechanisms that are ultimately linked by the PCr shuttle: 1) ATP levels and local ATP/ADP ratios in the cytosol are kept high, thus sustaining membrane ion gradients and other vital energy-consuming processes at a highly efficient level, and 2) mitochondria are protected from MPT pore opening via functional coupling of the mtCK reaction to oxidative phosphorylation.
Taken together, our findings offer important clues on MPT regulation by mitochondrial ADP phosphorylation and may have interesting implications for the design of creatine analogs to treat patients with neurodegenerative diseases. In order to fully exploit the advantages of the PCr shuttle for cell survival, such analogs would have to be substrates for CK in both the forward and reverse direction of the reaction.