Oxidative Stress, Thiol Reagents, and Membrane Potential Modulate the Mitochondrial Permeability Transition by Affecting Nucleotide Binding to the Adenine Nucleotide Translocase*

(Received for publication, May 17, 1996, and in revised form, October 31, 1996)

Andrew P. Halestrap Dagger , Kuei-Ying Woodfield and Cathal P. Connern

From the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 µM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 µM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 µM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.


INTRODUCTION

Mitochondria exposed to supraphysiological concentrations of [Ca2+] become nonselectively permeable to small molecules (<1500 Da). The sensitivity of this mitochondrial permeability transition (MPT)1 to [Ca2+] is greatly increased by oxidative stress, adenine nucleotide (AdN) depletion, elevated phosphate concentrations, low membrane potential (Delta psi ), and agents that stabilize the "c" conformation of the adenine nucleotide translocase (ANT). In contrast, protection is afforded by low pH, high Delta psi , and agents that stabilize the "m" conformation of the ANT (see Refs. 1-3). In addition, pore opening can be prevented by sub-micromolar concentrations of cyclosporin A (CsA) and its analogues (4-6). This effect appears to be mediated through the matrix peptidyl-proline cis-trans-isomerase, since the Ki values for inhibition of this enzyme by a number of CsA analogues correlate with their ability to inhibit pore opening (6-8). N-terminal sequencing of the purified peptidyl-prolyl cis-trans-isomerase has shown it to be a member of the cyclophilin (CyP) family (9) and most probably identical to the human CyP3 gene product (10), now more usually termed CyP-D (11, 12). These observations led us to suggest that an interaction of matrix CyP with an integral membrane protein leads to a change in its conformation which, when triggered by Ca2+, induces pore opening. In view of the influence of the ANT conformation on this process, we suggested that the integral membrane protein might be the ANT (6, 7). Further evidence in support of this model has come from our observations that oxidative stress, thiol reagents, NADH oxidation, increased matrix volume, and chaotropic agents, all of which enhance pore opening, also cause a recruitment of CyP to the inner mitochondrial membrane (13, 14). In contrast, modulation of pore opening by matrix pH, AdNs, Delta psi , and the conformation of the ANT appeared to be independent of CyP binding and must involve different regulatory sites on the pore complex (13, 14).

Matrix ADP is an important modulator of pore opening and acts by decreasing the sensitivity of the calcium trigger site to [Ca2+]. There are thought to be two ADP binding sites, one with high affinity associated with the ANT that is blocked by the inhibitor carboxyatractyloside (CAT) and another lower affinity site (15-18). Bernardi and colleagues (19-23) have provided convincing evidence that another important modulator of the MPT is Delta psi . Thus, at a constant intramitochondrial [Ca2+], progressive lowering of Delta psi by addition of uncoupler leads to a progressive stimulation of pore opening. Thiol reagents shift this voltage dependence, allowing the pore to open at higher Delta psi (19-23), implying that the voltage sensor possesses critical thiol groups whose modification attenuates the voltage sensitivity.

During our studies on the effects of thiol reagents on CyP binding, we noticed that phenylarsine oxide (PheArs) consistently produced a greater stimulation of pore opening than diamide or t-butylhydroperoxide (TBH), and yet induced less CyP binding. This led us to consider the possibility that thiol reagents might antagonize ADP inhibition of the MPT through an effect on the nucleotide binding site of the ANT, independent of their effects on CyP binding. Under the influence of a physiological Delta psi , the ANT drives ATP4- out of mitochondria in exchange for the entry of ADP3-. This electrically driven process is brought about by an effect of Delta psi on the conformation of the carrier and its affinity for AdNs at the extramitochondrial binding site (24-26). If Delta psi were also able to increase the affinity of the matrix facing nucleotide binding site for AdNs, this might explain the regulation of the pore by Delta psi . Thus at high Delta psi AdN binding would be tighter and the pore would be less sensitive to [Ca2+], while at lower Delta psi binding would become weaker and the sensitivity of the pore to [Ca2+] would increase. This effect would be exacerbated by thiol reagents if these decrease the affinity of AdN binding. This paper provides evidence in support of this hypothesis.


EXPERIMENTAL PROCEDURES

Materials

Rat liver mitochondria were prepared as described by Halestrap and Davidson (6). For some experiments, mitochondria were depleted of their endogenous AdNs by incubation for 15 min at 25 °C in sucrose isolation buffer supplemented with 2 mM PPi as described previously (27). N-terminal antipeptide antibodies to the mature mitochondrial cyclophilin were raised and purified as described elsewhere (13). Antibodies against bovine ANT and rat mitochondrial NAD+-dependent isocitrate dehydrogenase were kindly provided by Dr. Gerard Brandolin, Department of Molecular and Structural Biology, CNRS, F-38041 Grenoble, France, and Professor R. M. Denton of this department, respectively. Eosin 5-maleimide, safranin, 4-aminophenylarsine oxide, and deoxy-ADP were from Sigma. The sources of all other chemicals and biochemicals were as described previously (13, 14, 28).

Methods

Measurements of Pore Opening Mitochondrial Swelling

This was measured by the decrease in light scattering at 520 nm as described previously (28). Liver mitochondria were incubated under de-energized conditions at 25 °C and 0.5 mg of protein/ml in 3.5 ml of buffer containing 20 mM Mops, 10 mM Tris, 1 mM potassium phosphate, 2 mM nitrilotriacetic acid, 0.5 µM rotenone, 0.5 µM antimycin, 2 µM A23187, and 150 mM KCl, pH 7.2. The calcium ionophore A23187 was added to ensure complete equilibration of Ca2+ across the mitochondrial inner membrane under de-energized conditions (28). In experiments where the effects of ATP were to be studied, 0.5 µM oligomycin was also present. A520 was monitored continuously in a spectrophotometer with computerized data acquisition (2 data points per s) as described previously (6). When required, energization was achieved by addition of 2 mM succinate in the absence of antimycin and A23187; where indicated 150 mM KCl was replaced by 300 mM sucrose. Other additions were made as indicated in the figure legends. The effects of Delta psi and thiol reagents on pore opening in energized mitochondria were investigated using a procedure similar to that described by Petronilli et al. (22). Mitochondria (5 mg of protein) were added to 7 ml of buffer, and aliquots (3.5 ml) of this mitochondrial suspension were then added to both sample and reference cuvettes of a split-beam spectrophotometer (29), and A520 was monitored. After incubation at 25 °C for 1 min in the presence of succinate, pore opening was initiated by addition of Ca2+ (20-70 nmol/mg of protein) followed at 50-s intervals by 0.5 mM EGTA, the reagent of interest, and finally the uncoupler carbonyl cyanide p-trifluormethoxyphenylhydrazone (FCCP) at the required concentration.

Shrinkage of Pre-swollen Mitochondria

Mitochondria were pre-swollen as described previously (13, 14), by incubating for 20 min at 30 °C and 2 mg of protein/ml under de-energized conditions in KSCN buffer (150 mM KSCN, 20 mM Mops, 10 mM Tris, 0.5 µM rotenone, 0.5 µM antimycin at pH 7.2) containing 1 mM CaCl2. Additions of TBH, diamide, or PheArs to the buffer were made as required. Swelling was terminated by addition of 1.2 mM EGTA and centrifugation at 12,000 × g for 10 min to sediment the swollen mitochondria which were then resuspended at 20 mg of protein/ml in KSCN buffer containing 2 mM nitrilotriacetic acid and 2 µM A23187. Shrinkage of these pre-swollen mitochondria at different [Ca2+] was determined by measuring the rate of increase of A520 upon addition of 7% (w/v) polyethylene glycol (PEG) as described previously (13, 14). Briefly, pre-swollen mitochondria (approximately 1 mg of protein) were added to a cuvette containing 3 ml of KSCN buffer (with 2 mM nitrilotriacetic acid and 2 µM A23187) supplemented with CaCl2 to give the desired free [Ca2+] and other additions as required. After incubation for 1 min at 25 °C, measurement of A520 was initiated and monitored continuously (10 data points per s). Shrinkage was induced by addition of 0.5 ml of 50% (w/v) PEG 2000 through a light-sealed injection port followed by rapid mixing (<1 s). Shrinkage induced by the colloidal osmotic pressure of PEG was detected as a rapid increase in A520.

Confirmation of Mitochondrial Energization Using Safranin

Mitochondria (1 mg of protein/ml) were incubated in the cuvette of a double-beam spectrophotometer at 25 °C in buffer containing 125 mM KCl, 20 mM Mops, 10 mM Tris, 0.5 mM EGTA, 0.5 µM rotenone, and 10 µM safranin. Energization was determined as the difference in absorbance at 554 and 524 nm (A554-524) (30) and was monitored continuously. Energization was induced either by addition of 2 mM succinate or 0.2 mM ATP. Other additions were made as described in the figure legend. No attempt was made to calibrate Delta psi measurements.

Cyclophilin Binding

CyP binding to mitochondrial membranes was measured in parallel incubations as described previously (14). Mitochondrial membranes were prepared by freeze-thawing mitochondrial incubations following addition of KSCN buffer containing the protease inhibitors antipain, pepstatin, and leupeptin at 1 µg/ml and phenylmethylsulfonyl fluoride at 100 µM. Detection of CyP binding to the membranes was performed using SDS-PAGE (10 µg of protein per track determined by Bradford assay using bovine serum albumin as standard) and Western blotting with N-terminal antipeptide antibodies to mitochondrial CyP using an enhanced chemiluminescence detection kit (Amersham Corp., Bucks., UK). Western blotting with antibody to NAD+-dependent isocitrate dehydrogenase was performed in parallel to correct for changes in nonspecific CyP binding.

Binding of the ANT to a Phenylarsine Oxide Column

4-Aminophenylarsine oxide was conjugated to Affi-Gel 10 (Bio-Rad, prepared for reaction as described by the manufacturer). The 4-aminophenylarsine oxide was dissolved in H2O at 5 mg/ml by vigorous stirring and addition of 1 M HCl until the pH was 1.5. HEPES (50 mM) was then added and the pH brought to 7.2 with M NaOH. An aliquot equivalent to 3 mg of the reagent was reacted with 3 ml of Affi-Gel 10 overnight at room temperature, followed by blocking of unreacted groups by incubation for 1 h at pH 8.0 with 0.3 M ethanolamine. Columns (usually 0.5 ml) were poured and washed with 50 mM HEPES, pH 7.2, containing 150 mM Na2SO4, 1 mM EDTA, 0.25% (w/v) Triton X-100, and the protease inhibitors antipain, leupeptin, and pepstatin at 1 µg/ml and phenylmethylsulfonyl fluoride at 100 µM (Buffer A). Coupling efficiency of the phenylarsine oxide was not determined. Mitochondria were solubilized at 0 °C and 10 mg of protein per ml in Buffer A containing additional Triton X-100 (3%). Insoluble material was removed by centrifugation at 180,000 × g for 1 min in a Beckman Airfuge, and 1 ml of sample was added to a 0.5-ml column. Unbound material was washed through the column with Buffer A until no protein was detected in the effluent. Bound protein was then eluted with Buffer A containing 10 mM dithiothreitol, and the protein concentration in each fraction (0.6 ml) was determined by Bradford assay using bovine serum albumin as standard. Samples (20 µl) of both flow-through and eluted fractions were separated by SDS-PAGE on 15% acrylamide gels, and Western blotting was performed with anti-ANT antibodies.

Expression of Results

All light scattering traces shown are representative of at least three separate experiments. Initial rates were determined by differentiation of traces (ignoring the response during the first second which is associated with mixing artifacts) and used to provide a relative measure of the extent of mitochondrial pore opening. The resulting data were analyzed using FigP and PFit software (Biosoft, Cambridge, UK). The dependence of pore opening on [Ca2+] is shown as smooth curves generated by least squares regression analysis to Equation 1 for cooperative kinetics:
v=V<SUB><UP>min</UP></SUB>+(V<SUB><UP>max</UP></SUB>−V<SUB><UP>min</UP></SUB>)/(1+(([<UP>Ca</UP>]/K<SUB>0.5</SUB>)<SUP><UP>−</UP>n</SUP>)) (Eq. 1)
where Vmax and Vmin are the rates of shrinkage at maximal and zero [Ca2+], respectively, K0.5 is the [Ca2+] giving the half-maximal rate of shrinkage, and n is the Hill coefficient. It should be noted that the 1-s mixing time limited the maximum rates of light scattering changes that could be measured accurately to rates with a t0.5 of 5 s or greater. No attempt was made to determine rates at saturating [Ca2+], and thus, without an accurate estimate of Vmax, the values of K0.5 and n cannot be determined with certainty. Nevertheless, the shape of each curve was reproducible from day to day, and the plots obtained readily illustrate changes in Ca2+ sensitivity of pore opening. The inhibition of shrinkage by ADP was usually analyzed by least squares regression analysis using Equation 2 which assumes only one binding site for ADP:
v=V<SUB><UP>min</UP></SUB>+(V<SUB><UP>max</UP></SUB>−V<SUB><UP>min</UP></SUB>)/(1+[<UP>ADP</UP>]/K<SUB>i</SUB>) (Eq. 2)
where Vmax is the initial rate of shrinkage in the absence of ADP, Vmin the rate in the absence of Ca2+, and Ki is the dissociation constant for ADP binding to its inhibitory site. Where two binding sites were suggested by the data, Equation 3 was used:
v=V<SUB><UP>min</UP></SUB>+V<SUB><UP>max1</UP></SUB>/(1+[<UP>ADP</UP>]/K<SUB>i1</SUB>)+V<SUB><UP>max2</UP></SUB>/(1+[<UP>ADP</UP>]/K<SUB>i2</SUB>) (Eq. 3)
where Ki1 and Ki2 are the dissociation constants for the two ADP binding sites, and Vmax 1 and Vmax 2 represent that part of the Ca2+-sensitive rate of shrinkage that is inhibited when ADP binding sites 1 and 2, respectively, are saturated.


RESULTS AND DISCUSSION

Lack of Correlation Between the Effects of Different Thiol Reagents on CyP Binding and MPT Opening Suggests More Than One Site of Action

TBH, diamide, and PheArs all increase CyP binding to the inner membrane of de-energized mitochondria, in parallel with their ability to increase the Ca2+ sensitivity of the MPT (8, 13, 14, 31). However, as illustrated by the data in Fig. 1, 20 µM PheArs consistently gave a greater stimulation of the MPT than diamide, while the increase in CyP binding was substantially less. These data suggested that PheArs must exert an effect on the MPT independent of CyP binding. One such site of action could be on the ability of matrix ADP to inhibit the MPT, because ADP, in contrast to CsA, does not appear to displace membrane-bound CyP (Fig. 2).


Fig. 1. The relative effects of oxidative stress and thiol reagents on the MPT and CyP binding in de-energized mitochondria. Liver mitochondria were incubated at 25 °C under de-energized conditions in KCl buffer (see "Methods") with or without 1 mM TBH, 0.1 mM diamide, or 20 µM PheArs. A520 was continuously monitored and after 1 min CaCl2 was added to give the final buffered [Ca2+] of 150 µM. The inset shows Western blots of CyP binding to mitochondrial membranes prepared from parallel incubations of mitochondria. Track 1 represents membranes from control mitochondria (little or no bound CyP visible), and tracks 2-5 represents membranes from mitochondria pretreated with 1 mM TBH, 0.1 mM diamide, 20 µM PheArs, and 100 µM PheArs, respectively.
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Fig. 2. The effects of ADP and CsA on the Ca2+ sensitivity of the MPT pore. The extent of pore opening in pre-swollen mitochondria was measured at varying [Ca2+] using the rate of shrinkage induced by addition of 7% (w/v) PEG as described under "Methods." Where indicated 1 µM CsA, 20 µM ADP, 1 µM CsA + 20 µMADP, or 100 µM ADP were added 1 min before PEG addition. The inset shows Western blots of CyP and NAD+-dependent isocitrate dehydrogenase (ICDH) binding to mitochondrial membranes prepared from parallel incubations. Tracks represent membranes from pre-swollen mitochondria incubated for 1 min in the absence (track 1) or presence of 1 µM CsA (track 2), 0.2 mM ADP (track 3), or both CsA and ADP (track 4).
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In order to investigate the interaction of thiol reagents with ADP inhibition of the MPT, we used the PEG-induced shrinkage assay of the MPT first introduced by Haworth and Hunter (16). The advantage of this assay is that it allows the extent of pore opening to be determined after equilibration with defined matrix [Ca2+] and [ADP]. The data of Fig. 2 investigate the interaction between ADP and CsA on the Ca2+ sensitivity of pore opening in parallel with measurements of CyP binding. The data confirm that activation by Ca2+ is cooperative (16, 17) and that ADP and CsA both decrease the Ca2+ sensitivity by shifting the activation curve to the right. Comparison of the effect of 20 and 100 µM ADP shows that higher concentrations of ADP shift the curve further to the right suggesting that ADP acts as a competitive inhibitor of the Ca2+ trigger site in agreement with the conclusions of others (17, 18). In contrast, increasing the concentration of CsA from 0.2 to 1 µM had almost no additional effect (data not shown). Furthermore, the effects of ADP and CsA were additive, confirming that they act on different sites as implied by the CyP binding experiments. It should be noted that complete removal of bound CyP by addition of CsA was never obtained. Although this may account for the CsA-insensitive pore opening seen at high [Ca2+], it is more likely that bound CyP is not essential for pore opening but rather enhances its Ca2+ sensitivity (8, 13, 14, 31).

Oxidative Stress and Thiol Reagents Decrease the Affinity of ADP for Its Inhibitory Site

In Fig. 3 we investigate the effects of pretreatment of mitochondria with TBH, diamide, and PheArs on the inhibition of the MPT by ADP at a fixed [Ca2+]. Since such pretreatment increases the sensitivity of the MPT to [Ca2+] (13, 14), we chose to fix the [Ca2+] at different concentrations for the control and pretreated mitochondria in order to give similar rates of shrinkage in the absence of ADP. However, we performed ADP titrations on control mitochondria at two different [Ca2+] in order to confirm that [Ca2+] did not influence the Ki for ADP. Data are shown in Fig. 3A. The Ki values derived using a single site model were 6.9 ± 1.0 and 8.1 ± 1.1 µM at 50 and 100 µM [Ca2+], respectively. Thus Ca2+ does not appear to compete with ADP for a common binding site. In contast, pretreatment of mitochondria with TBH, diamide, and PheArs all greatly increased the Ki for ADP, as shown in Fig. 3. The data of Fig. 3A show that 1 mM TBH and 0.1 mM diamide gave a large increase in Ki from 8.1 ± 1.1 to 42.9 ± 2.9 µM and 91.4 ± 11.1 µM, respectively, while the data of Fig. 3B, obtained with a different mitochondrial preparation, show that pretreatment with 20 µM PheArs or 1 mM TBH increased the Ki from 1.7 ± 0.4 to 491 ± 27 µM and 67.0 ± 9.7 µM, respectively. The higher Ki values obtained in the control experiments of Fig. 3A than those of Fig. 3B reflect a variability in this parameter which we believe to reflect oxidative stress during the pre-swelling of the mitochondria. The presence of 1 µM CsA significantly reduced the [Ca2+] sensitivity of the MTP, requiring a higher [Ca2+] to induce the same rate of shrinkage. However, CsA had no effect on the Ki for ADP in either control or pretreated mitochondria as shown by the dashed traces of Fig. 3B. This is consistent with the lack of any interaction between CsA and ADP on CyP binding (Fig. 2).


Fig. 3. The effects of oxidative stress and thiol reagents on inhibition of the MPT by ADP. A, the ADP sensitivity of mitochondria pre-swollen in the absence (bullet , black-triangle) or presence of 1 mM TBH (black-square) or 0.1 mM diamide (square ) were determined using the PEG-induced shrinkage assay with [Ca2+] fixed at 50 (black-triangle, black-square, square ) or 100 µM (bullet ). Data were fitted by nonlinear least squares regression analysis to the equation for inhibition by ADP binding to a single noncooperative binding site as described in the text. In control mitochondria the derived Ki values were 6.9 ± 1.0 and 8.1 ± 1.1 µM at 50 and 100 µM [Ca2+], respectively. For the TBH-treated and diamide-treated mitochondria the derived Ki values were 42.9 ± 2.9 and 91.4 ± 11.1 µM, respectively. B, mitochondria pre-swollen in the absence (bullet ) or presence of 20 µM PheArs (black-triangle, triangle ), 1 mM TBH (black-square, square ), or 1 µM CsA (triangle , square ) were used with [Ca2+] fixed at 50 µM (black-triangle, black-square), 80 µM (bullet ), or 85 µM (triangle , square ). For ease of presentation, rates of shrinkage have been expressed as a percentage of the rate in the absence of ADP derived by regression analysis of the data. The derived Ki values for control, TBH-treated, and PheArs-treated mitochondria in the absence of CsA (solid lines) were 1.7 ± 0.4, 67.0 ± 9.7, and 491 ± 27 µM, respectively, with corresponding maximal rates of shrinkage being 190 ± 12.9, 222 ± 10.0, and 160 ± 2.7 A520·min-1·104. In the presence of 1 µM CsA (dashed lines) the Ki values for TBH-treated and PheArs-treated mitochondria were 51.0 ± 5.8 and 529 ± 77 µM and maximal rates of swelling 244 ± 9.1 and 283 ± 12.2 A520·min-1·104.
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In order to ascertain whether the effects of the thiol reagents were dependent on the presence of any matrix component such as glutathione, the effects of adding TBH, diamide, and PheArs to pre-swollen mitochondria, which will have lost their matrix glutathione (32), were investigated. The data of Fig. 4 show that addition of 0.1 mM diamide produced a small increase in Ki from 9.1 ± 0.4 to 13.0 ± 0.8 µM, whereas 50 µM PheArs added directly to the shrinking assay gave a considerable increase in the Ki for ADP (131 ± 14 µM). TBH was without any effect under these conditions (data not shown). These data are in agreement with the observations of Chernyak and Bernardi (33) who showed that TBH exerts its effect on the PheArs-sensitive thiol group through oxidized glutathione, and it is likely that diamide acts largely through the same mechanism. Oxidized glutathione may oxidize two closely associated protein thiols to form a disulfide as argued by Petronilli et al. (22) but is also capable of forming adducts with membrane proteins under conditions of TBH-induced oxidative stress (34, 35). An additional effect of diamide and TBH might be to oxidize matrix thioredoxin and lipoamide which have been proposed to interact with membrane protein thiols (36). In contrast, PheArs, which attacks vicinal thiols on proteins (37), appears to be able to attack the pore component directly, without involvement of matrix components such as glutathione.


Fig. 4. The effects of diamide and phenylarsine oxide added directly to pre-swollen mitochondria on the affinity of ADP for its inhibitory site. The protocol was the same as that for Fig. 3 except that control pre-swollen mitochondria were employed and incubated for 1 min with 50 µM Ca2+ in the absence (bullet ) or presence of 0.1 mM diamide (square ) or 50 µM PheArs (black-square) before addition of ADP at the concentration shown. Shrinking was initiated 1 min later by addition of PEG. The derived Ki values were 9.1 ± 0.4, 13.0 ± 0.8, and 131 ± 14 µM, respectively.
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The ANT Is the Site of Action of Matrix ADP and Its Antagonism by Thiol Modification

The suggestion that the ANT might be involved in pore formation (6, 38, 39) has recently been given further strong support by the demonstration that the ANT reconstituted into proteoliposomes exhibits Ca2+-dependent channel activity that is blocked by ADP in the presence of bongrekic acid (BKA) (31). Previous data investigating the effects of carboxyatractyloside (CAT) on the ADP inhibition of the MPT implied that ADP acts at two inhibitory sites, one on the ANT and the other an additional matrix facing site with lower affinity (15, 17, 18). We have investigated these binding sites further using the PEG-induced shrinkage assay to measure the effects of BKA and CAT on Ki for ADP inhibition of the MPT at fixed [Ca2+] (65 µM). These two inhibitors trap the ANT in the c and m conformations, respectively (40). BKA was found to have no effect on the Ki for ADP nor on the sensitivity of the MPT to matrix [Ca2+] under these conditions (data not shown). In contrast CAT was found to antagonize the effect of ADP as described by others (15-18), and this effect was analyzed more carefully in the data of Fig. 5. The effect of CAT could not be increased by using higher concentrations of the inhibitor and was equally effective at 1 µM, implying very tight binding to its inhibitory site. When data were fitted by nonlinear least squares regression analysis assuming a single inhibitory site, a good fit was obtained only when CAT was present, with a Ki value (± S.E. for the fit shown) of 17.8 ± 1.4 µM. However, in the absence of CAT the fit was poor at higher ADP concentrations (dashed line), and a better fit was obtained when the data were refitted using a two-site model (solid line). Derived values for Ki1 and Ki2 were 0.63 ± 0.17 and 17.2 ± 12.8 µM, respectively, and although the Ki2 could not be determined with great accuracy by such a regression analysis, it would appear to be similar to the single Ki value determined in the presence of CAT. Indeed in three similar experiments we found that when the two-site equation was used with the high Ki value fixed to that obtained in the presence of CAT, a good fit to the data was consistently obtained. The mean values (± S.E.) of the single site Ki for ADP in the absence and presence of CAT were 1.9 ± 0.4 and 27.2 ± 5.2 µM, respectively, whereas the high affinity Ki for the two-site model (assuming the low affinity site to have the same Ki as that in the presence of CAT) was 1.2 ± 0.32 µM. Thus our data are consistent with there being two sites for ADP inhibition, a high affinity site on the ANT (Ki about 1 µM) which is only present in the m but not the c conformation of the carrier and a lower affinity site (Ki about 25 µM) that is independent of carrier conformation. Neither site appears to be sensitive to CsA since the inhibition curve was not affected by the presence or absence of 1 µM CsA (Fig. 3), although the concentration of [Ca2+] had to be raised from 65 to 100 µM to give the same rate of shrinking.


Fig. 5. The effects of carboxyatractyloside on the sensitivity of the MPT to [ADP]. The concentration dependence of the ADP inhibition of pore opening was measured at a fixed [Ca2+] (65 µM) in the absence (bullet ) or presence of 10 µM CAT (square ) or at 100 µM [Ca2+] in the presence of 1 µM CsA (open circle ). Data were fitted by nonlinear least squares regression analysis to the equation for inhibition by ADP binding to a single noncooperative binding site. The derived Ki values (± S.E. for the fit shown) were 17.8 ± 1.4 µM in the presence of CAT and 1.31 ± 0.14 µM (dashed line) in the absence of CAT. The solid line for the data in the absence of CAT represents the fit to a two-site model, with derived Ki values of 0.63 ± 0.17 and 17.2 ± 12.8 µM. Addition of BKA was without effect on the ADP inhibition curve (data not shown). The dotted line (black-triangle) represents the difference in the rate of shrinkage observed in the presence and absence of CAT at the same [ADP].
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The ANT has two ADP binding sites of high and low affinity, respectively (40, 41). In liver mitochondria ADP binding to the high affinity site has a Kd of 0.5 µM and is displaced by CAT (40, 42). The Kd of the low affinity site for ADP has not been reported for liver mitochondria, but in heart mitochondria it is 10-40 times greater than the Kd of the high affinity site. This would suggest that it might be about 20 µM, similar to the value measured for inhibition of pore opening. Thus ADP might be exerting its effect entirely through an interaction with the ANT but on two distinct sites. The presence of two such AdN binding sites is also consistent with the emerging model of the mechanism of the ANT, which proposes a sequential model with two internal and two external substrate binding sites, one high and one low affinity site on each side of the membrane (41, 43, 44).

We have investigated the specificity of the nucleotide binding site for inhibition of pore opening by investigating the concentration dependence of inhibition by a variety of nucleotides. Additional CaCl2 was added where necessary to correct for the chelation of Ca2+ by some nucleotides such as ATP. The derived Ki values for ADP, deoxy-ADP, and ATP (± S.E. for a single site model) were 1.7 ± 0.4, 23.2 ± 2.4, and 544 ± 75 µM, respectively. The ability of deoxy-ADP to inhibit the pore with a 10-15-fold lower affinity than ADP is similar to the difference in Km of the ANT for ADP and deoxy-ADP (40). At 1 mM AMP, GDP, GTP, NAD+, NADP+, NADH, and NADPH were all without significant effect on the pore, correlating with their lack of binding to the ANT (40). CAT did not appear to have a significant effect on the inhibition caused by deoxy-ADP (data not shown), in contrast to its ability to raise the apparent Ki for ADP by more than 10-fold. This may indicate that deoxy-ADP is binding only at the low affinity ADP binding site.

We have confirmed that the large difference in the inhibitory potency of matrix ADP and ATP is not an artifact of using pre-swollen mitochondria in KSCN medium. Mitochondria were incubated in KCl medium under de-energized conditions in the presence of oligomycin, and either 100 µM ADP or ATP was added before initiation of pore opening with 200 µM Ca2+. In the absence of Delta psi ATP will enter in exchange for ADP, both nucleotides moving down their concentration gradients, and matrix [ATP] will be high relative to [ADP]. In contrast, after addition of ADP matrix [ATP] will be very low relative to [ADP]. In the presence of ADP, little or no pore opening was observed, whereas rapid swelling occurred when ATP was present (data not shown). This confirms that ATP is a relatively ineffective inhibitor of the pore in comparison to ADP.

Evidence That PheArs Binds to Specific Cysteine Groups on the ANT

Our data suggest that PheArs decreases binding of ADP to the ANT, implying that this reagent must bind to specific thiol groups on the carrier. We have sought to demonstrate this directly by synthesizing a phenylarsine oxide affinity column (see "Methods") and investigating the binding of the ANT to it. The data of Fig. 6 confirm that such binding occurs and is prevented by pretreatment of mitochondria with 20 µM CAT or PheArs before membrane solubilization. Binding was also greatly reduced by pretreatment with 0.1 mM diamide or 1 mM TBH (data not shown), confirming that these two reagents can also attack the thiol group modified by PheArs. This would be predicted from their ability to lower the affinity of ADP for its inhibitory site.


Fig. 6. Binding of the adenine nucleotide translocase to a phenylarsine oxide column. Detergent-solubilized extracts of control mitochondria and those pretreated with 20 µM CAT or 100 µM PheArs were added to a phenylarsine oxide column and unbound protein washed off before eluting with buffer containing dithiothreitol as described under "Methods." Samples of the flow-through fraction and successive eluted fractions were run on SDS-PAGE followed by Western blotting with anti-ANT antibodies. Only the band corresponding to the ANT is shown in each case. Parallel measurements of protein showed the CAT did not influence the total amount of protein bound, whereas pretreatment with PherArs reduced the bound protein by about 50%.
[View Larger Version of this Image (40K GIF file)]


The ANT is known to have three reactive cysteines (Cys56, Cys159, and Cys256) that are all on putative matrix-facing loops and whose thiol groups show differential reactivity to various thiol reagents in a conformation-dependent manner (45-47). Cys159 can be selectively labeled in submitochondrial particles with the membrane-impermeant thiol reagent, eosin 5-maleimide, which also inhibits transport. Labeling is decreased in the presence of ADP (45), suggesting that this residue is at or close to an ADP binding site, consistent with previous studies using lysine-specific labeling reagents that identified Lys162 and Lys165 as being involved in substrate binding (48). Thus, Cys159 would seem to be a possible locus for the effects of thiol reagents that antagonize ADP inhibition of the mitochondrial pore. We have tested this possibility by investigating the ADP sensitivity of the MPT in mitochondria incubated with 40 µM eosin maleimide during the pre-swelling. The Ca2+-dependent pore opening in these mitochondria became extremely insensitive to ADP inhibition, with only about 20% inhibition being observed at 0.5 mM ADP (data not shown). Thus it would seem possible that Cys159 is the locus of action of thiol reagents that affect the ADP sensitivity of the MPT. In parallel experiments we attempted to investigate whether eosin maleimide had any effect on CyP binding to the inner membrane but found that this reagent led to the formation of protein aggregates during SDS-PAGE that prevented consistent quantitative Western blots.

Another thiol group is probably involved in CyP binding to the inner membrane, and this might be Cys56 of the ANT. Thus at low concentrations (<50 µM) N-ethylmaleimide (NEM) binds to Cys56 only when the carrier is in the m conformation (40, 49), whereas LeQuoc and LeQuoc (50) have demonstrated that oxidative stress decreases NEM binding to the ANT, implying that such treatment modifies Cys56. Indeed, Zwizinski and Schmid (51) have reported that exposure of cardiac mitochondria to oxidative stress leads to an inhibition of ANT activity and a slight decrease in its mobility on SDS-PAGE. More striking is the demonstration by Majima et al. (47) that treatment of sub-mitochondrial particles with copper o-phenanthroline cross-links the ANT dimer by forming a disulfide bridge between the Cys56 residues of the two monomers. This reagent causes the formation of a disulfide bridge between two adjacent thiols and thus may well reflect the action of TBH and diamide. Indeed, these reagents have been shown to cross-link proteins in mitochondrial membranes (52).

The Effects of the Mitochondrial Delta psi on the MPT Is Mediated through AdN Binding to the ANT

The mitochondrial Delta psi greatly increases the efflux of matrix ATP on the ANT in exchange for ADP. This electrophoretic transport must entail an effect of Delta psi on either AdN binding to the ANT or on the translocation cycle (24, 25, 53). An increase in ATP binding to the matrix nucleotide binding site of the ANT caused by increased Delta psi is likely (24), and this would be capable of explaining the inhibitory effect of Delta psi on the MPT. Such an effect might be reflected by the ability of ATP to inhibit the MPT more effectively in energized than de-energized mitochondria. In order to investigate this, manipulation of the matrix ATP/ADP ratio in energized mitochondria was necessary. In the absence of oligomycin, succinate is well known to establish a high Delta psi and an ATP/ADP ratio of about 3 (24). However, if oligomycin is added before addition of succinate, oxidative phosphorylation is prevented and ATP/ADP ratios are an order of magnitude lower (24). In Fig. 7 we show that when energized mitochondria were loaded with moderate amounts of Ca2+ (50 µM), in the absence of oligomycin (i.e. low [ADP]), a small amount of pore opening (swelling) was apparent which was enhanced by addition of increasing concentrations of uncoupler. In the presence of oligomycin (i.e. high [ADP]) less pore opening was observed, and the process was less sensitive to uncoupler. These differences were exaggerated at higher Ca2+ (80 µM) as shown in Fig. 7B. Our data could be explained if Delta psi enhances the binding of AdNs to the inhibitory site of the MPT pore, ATP binding being influenced more than that of ADP. This would be consistent with the conformation-dependent binding of AdNs to the ANT (24, 25, 53).


Fig. 7. Comparison of pore opening in energized mitochondria with high or low matrix ATP/ADP. Mitochondria were preincubated for 1 min in KCl medium in the absence (solid line) or presence (dotted line) of 0.5 µM oligomycin to deplete the matrix of ATP. Energization was then achieved by addition of 2 mM Tris succinate and either 50 µM (A) or 80 µM (B) Ca2+ added 1 min later as indicated. EGTA (0.5 mM) was added after an additional 50 s and then further additions as indicated.
[View Larger Version of this Image (16K GIF file)]


If the explanation above is correct, the MPT in mitochondria depleted of AdNs by treatment with PPi (27) should be insensitive to Delta psi . The data of Figs. 8 and 9 demonstrate that this is the case. It was necessary, first, to establish that PPi-treated mitochondria are able to maintain a high Delta psi in the presence of succinate. This was confirmed by monitoring Delta psi with safranin as shown in Fig. 8. Addition of succinate to the de-energized mitochondria gave a similar change in safranin signal for both control and PPi-treated mitochondria, implying the generation of a similar Delta psi in both. In contrast, addition of ATP to energize the mitochondria produced a much smaller change in safranin signal in the PPi-treated mitochondria. This would be predicted since ATP is unable to enter the mitochondria on the ANT except in exchange for ADP, whose concentration is greatly reduced when the mitochondria are depleted of endogenous AdNs.


Fig. 8. Energization of control and PPi-treated mitochondria by succinate and ATP. Changes in Delta psi of control (solid line) and PPi-treated (dotted line) mitochondria incubated in KCl medium were monitored using safranin as described under "Methods." Additions were made of 2 mM succinate, 0.1 mM ATP, 0.5 µM oligomycin, 0.5 µM antimycin, and 1 µM FCCP as indicated.
[View Larger Version of this Image (16K GIF file)]



Fig. 9. The effects of thiol reagents and oxidative stress in control and PPi-treated mitochondria on pore opening induced by membrane depolarization with uncoupler. A, control mitochondria were incubated in sucrose medium under energized conditions for 1 min before addition of 70 µM Ca2+ followed 50 s later by addition of EGTA, then, if required, by 1 mM TBH, 0.1 mM diamide, or 20 µM PheArs as indicated. Finally FCCP was added at 50 nM unless otherwise indicated. B, PPi-treated mitochondria were used and [Ca2+] was added at 30 µM. In the top two traces the protocol for control and diamide traces was the same as for A, whereas in the lower traces the thiol reagents were added 1 min before addition of Ca2+.
[View Larger Version of this Image (25K GIF file)]


In Fig. 9A we confirm the effects of thiol reagents on pore opening in control mitochondria induced by membrane depolarization with uncoupler. Mitochondria incubated with 70 µM Ca2+ alone did not demonstrate significant pore opening. However, addition of EGTA after 50 s followed by addition of uncoupler did initiate swelling, the extent of which was increased as the concentration of uncoupler was increased. As described by Bernardi and colleagues (19-23), the sensitivity toward uncoupler was greatly increased by the presence of diamide, TBH, or PheArs. In contrast, mitochondria that had been pretreated with PPi to deplete them of endogenous AdNs were insensitive to uncoupler whether or not they had been treated with thiol reagents (Fig. 9B). Furthermore, as expected for mitochondria whose matrix has been depleted of AdNs, sensitivity to [Ca2+] was very much greater. Thus swelling was apparent 30 s after addition of 30 µM Ca2+ and was not further stimulated by additions of EGTA and FCCP, implying that depolarization had no effect on the MPT under these conditions. Higher concentrations gave faster rates of swelling, again unstimulated by uncoupler (data not shown). In fact, in contrast to control mitochondria, it can be seen from the data of Fig. 9B that addition of EGTA and uncoupler to the PPi-treated mitochondria after calcium loading actually inhibited swelling. The data of Fig. 9B also demonstrate that TBH, diamide, and PheArs had little or no effect on the swelling of energized PPi-treated mitochondria. This is consistent with these reagents sensitizing the MPT to depolarization through an effect on the binding of matrix AdNs to the ANT; with little or no AdNs present in these mitochondria no sensitizing effect would be possible.

Conclusions

Our data imply that thiol reagents can increase the Ca2+ sensitivity of the MTP by two mechanisms: first through an increase in CyP binding to its membrane target protein on the pore complex, and second through a decrease in ADP binding to its inhibitory site(s). Both of these actions could be exerted on the same target protein, and the evidence discussed above continues to make the ANT an attractive candidate as suggested in our original model of pore opening (6, 7). The recent demonstration that the purified and reconstituted ANT can form Ca2+-dependent channels that are blocked by ADP and BKA provides additional strong evidence for this hypothesis (31). Our data also provide an explanation for the observed voltage dependence of the MPT and the ability of thiol reagents to allow pore opening at higher Delta psi (22, 23). We suggest that the ANT itself may be the voltage sensor, because its binding of matrix AdNs is Delta psi -dependent and antagonized by thiol group modification. We believe that our data allow the regulation of the MPT by a variety of effectors to be grouped into three broad categories: those that modify AdN binding to the ANT, those that affect CyP binding, and those that interact directly with the Ca2+ trigger site. These are summarized in Table I.

Table I.

Proposed sites of action of known modulators of the mitochondrial permeability transition

The site of action of the different modulators is that which we propose in the text where the detailed rationale is given and references cited. Note that both CyP binding and ADP binding exert their effects through changes in the sensitivity of the MPT to [Ca2+].
Effect via change in cyclophilin binding Effect via change in nucleotide binding Direct effect on Ca2+ binding

Activatory
  Thiol reagents (e.g. diamide PheArs) Thiol reagents (e.g. diamide PheArs) High pH
  Oxidative stress (e.g. TBH) Oxidative stress (e.g. TBH)
  Increased matrix volume "C" conformation of ANT
  Chaotropic agents
Inhibitory
  CsA Membrane potential Low pH
"M" conformation of ANT Mg2+

An involvement of the unique mitochondrial CyP-D (9, 10) is now widely accepted and accounts for the ability of CsA analogues to inhibit pore opening with the same affinity as they inhibit the peptidyl-prolyl cis-trans-isomerase activity of CyP-D (7, 8, 54). We have shown that CyP-D binding to its membrane target protein is increased by a variety of procedures that also sensitize pore opening to [Ca2+] including oxidation of matrix NADH, oxidative stress induced by TBH, thiol reagents, increased matrix volume, and chaotropic agents (13, 14). As outlined above, we suggest that Cys56 may be the thiol group whose modification is responsible for the increased CyP binding by thiol reagents. This is close to Pro61 that we suggested in our original model of pore opening might bind CyP (6, 7). In this context, it may be significant that using a wide range of conditions we have been unable to demonstrate the MPT is mitochondria from yeast (Saccharomyces cerevisiae), and the three ANT isoforms of these mitochondria all lack Cys56 and Pro61 both of which are conserved in mammalian mitochondrial ANT isoforms (55, 56).

Our original model for the mechanism of pore formation suggested that mitochondrial CyP binding to the ANT in the presence of Ca2+ might induce a conformational change sufficient to form the pore (6, 7). However, on the current balance of data, it seems likely that the pore may be able to open in the absence of CyP binding but that this requires substantially higher [Ca2+]. Thus studies on the megachannel of patched clamped mitochondria, which probably reflects pore opening, have shown that the inhibitory effect of CsA is overcome at higher [Ca2+] (57, 58). Studies of Novgorodov et al. (17), Crompton and Andreeva (59), and ourselves (Fig. 2 and Ref. 17), using different techniques, have shown that at high matrix [Ca2+] inhibition of the MPT by CsA is overcome, especially in the absence of matrix AdNs. Finally, there is the recent demonstration that the purified and reconstituted ANT can form Ca2+-dependent channels resembling the permeability transition pore (31). Thus the evidence favors a modulatory role of CyP on pore opening rather than an essential role.

The regulation of the MPT by matrix AdNs, the conformation of the ANT, and Delta psi were all without effect on CyP-D recruitment to the membrane (Fig. 2 and Ref. 14), implying an alternative mode of action for these effectors. The data we present here suggest that the ANT provides a common locus for these effects. Binding of matrix AdNs to the ANT inhibits pore formation, and this binding is antagonized by thiol reagents, the c conformation of the carrier, and membrane depolarization. In contrast the m conformation and increased Delta psi enhance binding and inhibit the pore. As oulined above, we suggest that Cys159 may be the thiol group responsible for the antagonistic effects of thiol reagents on ADP inhibition of the pore. The binding of ADP to its regulatory site(s) does not appear to be influenced by [Ca2+] (Fig. 3A) suggesting that there is not direct competition between ADP and Ca2+ but rather that ADP causes the interconversion of the Ca2+-binding component of the pore from a high affinity to a low affinity conformation. In contrast to Novgorodov et al. (17), we find no evidence for CsA influencing the affinity of ADP binding to its inhibitory site(s) (Fig. 3B). Rather its effect to desensitize the pore to [Ca2+] appears to be additive with respect to ADP (Fig. 2) suggesting an alternative site of action. Thus our data are compatible with ADP and CyP binding to distinct sites on the ANT and inducing, respectively, a decrease or increase in the affinity of Ca2+ binding to the site which triggers pore opening. We suggest that the inhibitory effects of Mg2+ and H+ (low pH) are through direct competition with Ca2+ at its binding site (14, 16, 28, 60). Calmodulin does not appear to be associated with the Ca2+-binding site since the calmodulin antagonist trifluoperazine (1-100 µM) gave no inhibition of the rate of shrinkage of pre-swollen mitochondria (data not shown).

An association of the mitochondrial benzodiazepine receptor with porin and the ANT has been reported (61), and the possibility that this might represent the pore complex or some part of it is attractive (see Ref. 3). However, using the shrinkage assay we failed to detect any effect of diazepam or protopophyrin IX, both ligands for the mitochondrial benzodiazepine receptor (62), over a range of concentrations (50 nM to 50 µM).


FOOTNOTES

*   This work was supported by The Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 44 117 928 8592; Fax: 44 117 928 8274; E-mail: a.halestrap{at}bristol.ac.uk.
1    The abbreviations used are: MPT, mitochondrial permeability transition; ANT, adenine nucleotide translocase; AdN, adenine nucleotide; BKA, bongkrekate; CAT, carboxyatractyloside; PEG, polyethylene glycol; CyP, cyclophilin; PheArs, phenylarsine oxide; CsA, cyclosporin A; TBH, t-butyl hydroperoxide; Delta psi , mitochondrial membrane potential; FCCP, carbonyl cyanide p-trifluormethoxyphenylhydrazone; PAGE, polyacrylamide gel electrophoresis; Mops, 4-morpholinepropanesulfonic acid.

Acknowledgment

We thank Dr. Gerard Brandolin for the generous gift of antibodies against bovine ANT.


REFERENCES

  1. Bernardi, P., Broekemeier, K. M., and Pfeiffer, D. R. (1994) J. Bioenerg. Biomembr. 26, 509-517 [Medline] [Order article via Infotrieve]
  2. Halestrap, A. P. (1994) in Mitochondria: DNA, Proteins and Disease (Darley-Usmar, V., and Schapira, A. H. V., eds), pp. 113-142, Portland Press Ltd., London
  3. Zoratti, M., and Szabo, I. (1995) Biochim. Biophys. Acta 1241, 139-176 [Medline] [Order article via Infotrieve]
  4. Crompton, M., Ellinger, H., and Costi, A. (1988) Biochem. J. 255, 357-360 [Medline] [Order article via Infotrieve]
  5. Broekemeier, K. M., Dempsey, M. E., and Pfeiffer, D. R. (1989) J. Biol. Chem. 264, 7826-7830 [Abstract/Free Full Text]
  6. Halestrap, A. P., and Davidson, A. M. (1990) Biochem. J. 268, 153-160 [Medline] [Order article via Infotrieve]
  7. Griffiths, E. J., and Halestrap, A. P. (1995) Biochem. J. 307, 93-98 [Medline] [Order article via Infotrieve]
  8. Nicolli, A., Basso, E., Petronilli, V., Wenger, R. M., and Bernardi, P. (1996) J. Biol. Chem. 271, 2185-2192 [Abstract/Free Full Text]
  9. Connern, C. P., and Halestrap, A. P. (1992) Biochem. J. 284, 381-385 [Medline] [Order article via Infotrieve]
  10. Bergsma, D. J., Eder, C., Gross, M., Kersten, H., Sylvester, D., Appelbaum, E., Cusimano, D., Livi, G. P., McLaughlin, M. M., Kasyan, K., Porter, T. G., Silverman, C., Dunnington, D., Hand, A., Prichett, W. P., Bossard, M. J., Brandt, M., and Levy, M. A. (1991) J. Biol. Chem. 266, 23204-23214 [Abstract/Free Full Text]
  11. Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1994) FASEB J. 8, 391-400 [Abstract/Free Full Text]
  12. Galat, A., and Metcalfe, S. M. (1995) Prog. Biophys. Mol. Biol. 63, 67-118 [CrossRef][Medline] [Order article via Infotrieve]
  13. Connern, C. P., and Halestrap, A. P. (1994) Biochem. J. 302, 321-324 [Medline] [Order article via Infotrieve]
  14. Connern, C. P., and Halestrap, A. P. (1996) Biochemistry 35, 8172-8180 [CrossRef][Medline] [Order article via Infotrieve]
  15. Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys. 195, 453-459 [Medline] [Order article via Infotrieve]
  16. Haworth, R. A., and Hunter, D. S. (1979) Arch. Biochem. Biophys. 195, 460-467 [Medline] [Order article via Infotrieve]
  17. Novgorodov, S. A., Gudz, T. I., Milgrom, Y. M., and Brierley, G. P. (1992) J. Biol. Chem. 267, 16274-16282 [Abstract/Free Full Text]
  18. Novgorodov, S. A., Gudz, T. I., Brierley, G. P., and Pfeiffer, D. R. (1994) Arch. Biochem. Biophys. 311, 219-228 [CrossRef][Medline] [Order article via Infotrieve]
  19. Bernardi, P. (1992) J. Biol. Chem. 267, 8834-8839 [Abstract/Free Full Text]
  20. Petronilli, V., Cola, C., and Bernardi, P. (1993) J. Biol. Chem. 268, 1011-1016 [Abstract/Free Full Text]
  21. Petronilli, V., Cola, C., Massari, S., Colonna, R., and Bernardi, P. (1993) J. Biol. Chem. 268, 21939-21945 [Abstract/Free Full Text]
  22. Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642 [Abstract/Free Full Text]
  23. Petronilli, V., Nicolli, A., Costantini, P., Colonna, R., and Bernardi, P. (1994) Biochim. Biophys. Acta 1187, 255-259 [Medline] [Order article via Infotrieve]
  24. Souverijn, J. H. M., Huisman, L. A., Rosing, J., and Kemp, N. (1973) Biochim. Biophys. Acta 305, 185-198 [Medline] [Order article via Infotrieve]
  25. Klingenberg, M. (1980) J. Membr. Biol. 56, 97-105 [Medline] [Order article via Infotrieve]
  26. Krämer, R., and Klingenberg, M. (1982) Biochemistry 21, 1082-1089 [Medline] [Order article via Infotrieve]
  27. Griffiths, E. J., and Halestrap, A. P. (1991) Biochem. J. 274, 611-614 [Medline] [Order article via Infotrieve]
  28. Halestrap, A. P. (1991) Biochem. J. 278, 715-719 [Medline] [Order article via Infotrieve]
  29. Davidson, A. M., and Halestrap, A. P. (1990) Biochem. J. 268, 147-152 [Medline] [Order article via Infotrieve]
  30. Åkerman, K. E. O., and Wikström, M. K. F (1976) FEBS Lett. 68, 191-197 [CrossRef][Medline] [Order article via Infotrieve]
  31. Brustovetsky, N., and Klingenberg, M. (1996) Biochemistry 35, 8483-8488 [CrossRef][Medline] [Order article via Infotrieve]
  32. Savage, M. K., Jones, D. P., and Reed, D. J. (1991) Arch. Biochem. Biophys. 290, 51-56 [Medline] [Order article via Infotrieve]
  33. Chernyak, B. V., and Bernardi, P. (1996) Eur. J. Biochem. 238, 623-630 [Abstract]
  34. Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993) Arch. Biochem. Biophys. 307, 1-7 [CrossRef][Medline] [Order article via Infotrieve]
  35. Chai, Y. C., Hendrich, S., and Thomas, J. A. (1994) Arch. Biochem. Biophys. 310, 264-272 [CrossRef][Medline] [Order article via Infotrieve]
  36. Rigobello, M. P., Turcato, F., and Bindoli, A. (1995) Arch. Biochem. Biophys. 319, 225-230 [CrossRef][Medline] [Order article via Infotrieve]
  37. Zahler, W. M., and Cleland, W. W. (1968) J. Biol. Chem. 243, 716-719 [Abstract/Free Full Text]
  38. LeQuoc, K., and LeQuoc, D. (1988) Arch. Biochem. Biophys. 265, 249-257 [Medline] [Order article via Infotrieve]
  39. Macedo, D. V., Nepomuceno, M. D., and Pereiradasilva, L. (1993) Eur. J. Biochem. 215, 595-600 [Abstract]
  40. Klingenberg, M. (1976) in The Enzymes of Biological Membranes (Martonosi, A., ed), Vol. 3, pp. 383-483, Plenum Publishing Corp., New York
  41. Vignais, P. V., Brandolin, G., Boulay, F., Dalbon, P., Block, M., and Gauche, I. (1989) in Anion Carriers of Mitochondrial Membranes (Azzi, A., Fonyo, A., Nalecz, M. J., Vignais, P. V., and Wojtcak, L., eds), pp. 133-146, Springer Verlag, Berlin
  42. Klingenberg, M., Appel, M., Babel, W., and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654 [Abstract]
  43. Sluse, F. E., Sluse-Goffart, C. M., and Duyckaerts, C. (1989) in Anion Carriers of Mitochondrial Membranes (Azzi, A., Fonyo, A., Nalecz, M. J., Vignais, P. V., and Wojtcak, L., eds), pp. 183-195, Springer Verlag, Berlin
  44. Brandolin, G., Marty, I., and Vignais, P. V. (1990) Biochemistry 29, 9720-9727 [Medline] [Order article via Infotrieve]
  45. Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187 [Abstract/Free Full Text]
  46. Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536 [Medline] [Order article via Infotrieve]
  47. Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol. Chem. 270, 29548-29554 [Abstract/Free Full Text]
  48. Bogner, W., Aquila, H., and Klingenberg, M. (1986) Eur. J. Biochem. 161, 611-620 [Abstract]
  49. Boulay, F., and Vignais, P. V. (1984) Biochemistry 23, 4807-4812 [Medline] [Order article via Infotrieve]
  50. LeQuoc, D., and LeQuoc, K. (1989) Arch. Biochem. Biophys. 273, 466-478 [Medline] [Order article via Infotrieve]
  51. Zwizinski, C. W., and Schmid, H. H. O. (1992) Arch. Biochem. Biophys. 294, 178-183 [Medline] [Order article via Infotrieve]
  52. Fagian, M. M., Pereira-da-Silva, L., Martins, I. S., and Vercesi, A. E. (1990) J. Biol. Chem. 265, 19955-19960 [Abstract/Free Full Text]
  53. Krämer, R., and Klingenberg, M. (1982) Biochemistry 21, 1082-1089 [Medline] [Order article via Infotrieve]
  54. Tanveer, A., Virji, S., Andreeva, L., Totty, N. F., Hsuan, J. J., Ward, J. M., and Crompton, M. (1996) Eur. J. Biochem. 238, 166-172 [Abstract]
  55. Kolarov, J., Kolarova, N., and Nelson, N. (1990) J. Biol. Chem. 265, 12711-12716 [Abstract/Free Full Text]
  56. Cozens, A. L., Runswick, M. J., and Walker, J. E. (1989) J. Mol. Biol. 206, 261-280 [Medline] [Order article via Infotrieve]
  57. Szabo, I., and Zoratti, M. (1991) J. Biol. Chem. 266, 3376-3379 [Abstract/Free Full Text]
  58. Zoratti, M., and Szabo, I. (1994) J. Bioenerg. Biomembr. 26, 543-553 [Medline] [Order article via Infotrieve]
  59. Crompton, M., and Andreeva, L. (1994) Biochem. J. 302, 181-185 [Medline] [Order article via Infotrieve]
  60. Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabo, I., and Zoratti, M. (1992) J. Biol. Chem. 267, 2934-2939 [Abstract/Free Full Text]
  61. McEnery, M. W., Snowman, A. M., Trifiletti, R. R., and Snyder, S. H. (1992) Proc. Nat. Acad. Sci. U. S. A. 89, 3170-3174 [Abstract]
  62. Krueger, K. E. (1995) Biochim. Biophys. Acta 1241, 453-470 [Medline] [Order article via Infotrieve]

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