©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Voltage-dependent Porin-like Channel in the Inner Envelope Membrane of Plant Chloroplasts (*)

Bruno Fuks (§) , Fabrice Homblé (¶)

From the (1) Laboratoire de Physiologie Végétale, Faculté des Sciences C. P. 206/2, Université Libre de Bruxelles, Boulevard du Triomphe, B-1050 Brussels, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The electrical activity of a single channel of 525 ± 12 picosiemens in 150 mM KCl was measured after fusion of the inner envelope membrane of the chloroplast with planar lipid bilayers. The reversal potentials measured in KCl gradients indicate that this channel is weakly anion selective ( P/ P= 1.6 ± 0.2). The gating mechanism of the pore is voltage dependent. The channel shifts from a fully open state to a substate at positive electrical potentials and remains closed at negative electrical potentials. Succinylation of the protein increases the open probability of the fully open state and reverses the channel selectivity. Analysis of the single-channel conductance as a function of the salt concentration and of the open probability at various voltages suggests that this channel is a new membrane porin not previously identified.


INTRODUCTION

The exchange of solutes between the stroma of the chloroplast and the cytoplasm proceeds through the envelope made of the inner and the outer membrane. The outer envelope membrane of intact chloroplasts has been shown to be freely permeable to molecules of several kDa (1) . A pore-like channel has been identified in the outer envelope membrane of spinach chloroplast after detergent-solubilized membranes were fused with planar lipid bilayers. Its conductance of 720 pS() (in 100 mM KCl) was assumed to be responsible for the high permeability of this membrane (2) . Three other channels have been identified in the outer membrane of giant chloroplasts of Nitella (3) : two are cation selective channels with a conductance of 520 and 1 nS (in 100 mM KCl) and one 160 pS channel is anion selective. The voltage dependence and the conductance of the three large channels are similar to that of the mitochondrial porin, also called the voltage-dependent anion channel of the mitochondrial outer membrane (VDAC) (4) .

The inner envelope membrane of chloroplasts is much more selective; it contains a H-ATPase (5) and several specific carrier proteins. Two of them have been purified: the phosphate translocator (6) and the glycolate transporter (7) . Little is known about ion channels in the inner envelope membrane. The pH gradient across the inner envelope membrane is thought to be regulated by an active extrusion of Hbalanced by a passive counterflux through Hand Kchannels (8, 9) . An inner envelope Kchannel has been recently reconstituted into a planar lipid bilayer (10) . This channel could be involved in the pH regulation of the stroma. An anionic flux through the inner envelope membrane was also detected from swelling studies (11) and tracer flux experiments (12) done with intact chloroplastic suspensions. The chloride transporter could be the phosphate translocator, which can also work as an anion channel (13) .

We describe here the molecular properties of a new porin-like channel that was detected after fusion of the inner envelope membrane of chloroplasts with a planar lipid bilayer.


EXPERIMENTAL PROCEDURES

Purification of the Inner Envelope Membrane

Inner envelope membranes of chloroplast were purified from Spinacia oleracia by step-gradient centrifugation as described by Keegstra and Yousif (14) . Spinach leaves were purchased from a local market. About 500 g were homogenized with a Waring blender in 1 liter of grinding medium (GM) (0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl, 50 mM Tricine-KOH, pH 7.8, and 0.1% bovine serum albumin). All operations were carried out at 4 °C in a cold room. The mixture was filtered through eight layers of cheese cloth and one layer of cotton wool, and then 4 250 ml were centrifuged at 2,500 g for 2 min (Sorvall GSA rotor). The pellet was collected, resuspended in 2 10 ml of GM, and layered over 2 15 ml of 40% (v/v) Percoll (Pharmacia Biotech Inc.) in GM. The two tubes were centrifuged at 2,000 g for 2.5 min and then brought to rest without braking (Heraeus, swinging buckets). The broken chloroplasts and contaminant organelles that formed a band above the pellet were discarded. Purified intact chloroplasts were recovered at the bottom of the tube and washed two times in GM without bovine serum albumin at 2,700 g for 7 min (Sigma centrifuge). The integrity of the chloroplasts was higher than 95%, as determined with a Clark-type oxygen electrode (15) . Organelles (25 mg of chlorophyll) were resuspended in hypertonic medium (0.6 M sucrose, 2 mM EDTA, and 10 mM Tricine-KOH (pH 7.8)) and were incubated on ice for 15-20 min at 1 mg/ml chlorophyll. Then, inner and outer envelopes were detached by 30 strokes with a Dounce homogeneizer. The mixture was diluted with two volumes of 2 mM EDTA and 10 mM Tricine-KOH (pH 7.8) to give 0.2 M sucrose (final). Thylakoids and whole intact chloroplasts were removed by a centrifugation at 6,000 g for 10 min (Sorvall SS34 rotor). The yellow supernatant was collected and pelleted at 40,000 g for 30 min (Sorvall SS34 rotor) to obtain the crude envelope. The pellet was suspended in 2 20 ml of 0.2 M sucrose, 2 mM EDTA, and 10 mM Tricine-KOH (pH 7.8) and layered on the top of a discontinuous sucrose gradient (0.46 M (1.06 g/ml), 0.8 M (1.10 g/ml), and 1 M sucrose (1.13 g/ml)) in 2 mM EDTA, 10 mM Tricine-KOH (pH 7.8) and centrifuged at 120,000 g overnight (Beckman SW27 rotor). Outer and inner envelope fractions formed two bands, which were recovered separately at the second and third interfaces of the sucrose gradient, respectively. The outer and inner envelopes were diluted four times with 2 mM EDTA, 10 mM Tricine-KOH (pH 7.8) and were collected after a centrifugation of 125,000 g for 90 min (Beckmann SW27 rotor). The two pellets were resuspended with a Dounce homogeneizer in GM. The absence of cytochrome c oxidase activity and chlorophyll indicated that there was no detectable contamination by mitochondrial membranes and thylakoids. Small aliquots were stored at 70 °C. Intact chloroplasts equivalent to 1 mg of chlorophyll yielded about 10 µg of inner envelope proteins and about 5 µg of outer envelope proteins. The experiments reported in this paper were carried out using five batches of membrane purified during one year.

Electrical Measurements in Planar Lipid Bilayers

Planar lipid membranes were formed using the Mueller-Rudin technique (16) from a binary mixture of POPE/POPS (7:3 (w/w), Avanti Polar Lipids, AL) dissolved in n-decane. Experiments were performed at room temperature. All solutions of triple distilled water were millipore filtered. The two chambers were connected to the voltage generator through Ag/AgCl electrodes and a bridge of 3 M KCl, 2% agar. The current was measured using a Biologic RK-300 patch-clamp amplifier (Claix, France) and stored in a digital form on a videotape. The data were filtered with a low-pass 5-pole Tchebicheff filter at a cut-off frequency of 300 Hz. Acquired data were subsequently replayed, and the relevent events were sampled at 1 kHz and stored in a 386-Olivetti computer. Electrical potentials were defined as cis with respect to trans, which was held at ground.

The amplitude of the single-channel current was determined from total amplitude histograms fitted with Gaussian functions. The open probability of the fully open state ( P) was calculated according to the equation (17) P= (1/ N) P, where is the summation index from 0 to N, Pis the fraction of time during which a channel opens, and Nis the total number of channels in the planar lipid bilayer. In the absence of a substate, the channel behaves like a two-state channel. In this case, the open probability was estimated from records containing one or two channels. In contrast, in the presence of the substate, the open probability of the fully open state and the open probability of the substate were estimated from the fraction of time spent in the fully open state or in the substate. In this case, experiments containing only one channel were used for the analysis.

An aliquot of the inner envelope (5-10 µg of proteins) was sonicated for 20 s and added to the cis chamber under continuous stirring. Fusion of vesicles required a large osmotic gradient across the bilayer (cis side: 150 mM KCl, 5 mM CaCl, 10 mM Hepes-KOH, pH 7.2; trans side: 10 mM Hepes-KOH, pH 7.2) and an electrical potential of +60 mV across the bilayer. After fusion, an aliquot of 3 M KCl was added in the trans side (150 mM KCl, final concentration).

The conductivity of the experimental salt solution was measured using a Philips digital conductivity meter.

In the text, data are expressed as mean ± S.E. with the number of experiments in brackets. Statistical significance refers to a Student's t test ( p < 0.05).

Chemical Modification

Succinic anhydride (Sigma) was added from a stock solution of dimethyl sulfoxide to both cis and trans compartments to a final concentration of 3.8 mM. The compartments were stirred during 30 s, and measurements were performed after waiting for 5 min of incubation for a complete hydrolysis of succinic anhydride (the anhydride half-life is about 2 min). No electrical potential was applied across the bilayer during the incubation of the ion channel with succinic anhydride. Control experiments showed that the conductances of pure lipid bilayers were not modified by MeSO alone or 3.8 mM succinic anhydride, but addition of higher concentration of anhydride destabilized the planar bilayer. We used a 150 mM KCl solution buffered with 50 mM MOPS-KOH, pH 7.2, to prevent a pH decrease during hydrolysis of succinic anhydride, which could affect the anhydride reaction with amino groups (18) . In experiments carried out in a 1 M, 0.1 M KCl gradient, no succinic anhydride was added in the low salt compartment (the trans side) to minimize increase of salt buffer due to the presence of MOPS.

Dextran sulfate (8 kDa) (Sigma) was added to both compartments under continuous stirring for 1 min. Two concentrations were used: 6.3 and 31 µM (final concentrations). At each dextran concentration, various voltages were applied to determine the channel conductance and the open probability.


RESULTS

Conductance and Selectivity of the Channel

Isolated inner envelope membrane of chloroplasts are incorporated into a planar lipid bilayer separating two aqueous solutions. After addition of membrane vesicles to the cis side of the planar lipid bilayer and stirring for a maximum of 30 min, we sometimes observed a discrete jump of currents in the electrical record reflecting fusion between a vesicle containing one channel and the planar bilayer. Channels were successfully reconstituted in about 20% out of the trials. One channel of large conductance was reliably observed in planar bilayers enriched with the inner envelope. This channel was detected in 63 out of 84 successful reconstitutions of the inner envelope fractions. It is quite specific of the inner envelope and was never observed when the reconstitution was carried out with the thylakoids ( n > 50) or the outer envelope fraction ( n > 20). The channel remained mainly closed at negative potentials (Fig. 1). At positive potentials, the channel opened more frequently. Besides the transition between a fully open and a fully closed state, the channel shifted from its open state to a substate at +50 and +60 mV. Fig. 2 A shows the I-V relationship of the fully open state in symmetrical 150 mM KCl. A slope conductance of 525 ± 12 pS ( n = 26) is calculated between ±40 mV from the linear regression of the ohmic part of the curve. The current characterizing the substate is about half that the maximum opening (Fig. 2 B).


Figure 1: Typical current fluctuations of a single channel from the inner envelope of chloroplasts. The inner envelope membrane was incorporated into a planar lipid bilayer of POPE/POPS (7:3, w/w). The electrical measurements were carried out in symmetrical solution (150 mM KCl, 10 mM Hepes-KOH (pH 7.2)). The electrical potential difference (mV) is indicated at the right of each current trace. The closed state is indicated by c. Positive currents flowing from the cis to the trans side are plotted downward. The arrows indicate the substate of the channel.




Figure 2: I-V relationships of the channel. A, the I-V relationship was measured in symmetrical conditions (150 mM KCl, 10 mM Hepes-KOH (pH 7.2)). The data from 26 independent experiments were fitted by a third-order polynomial regression ( r= 1). The slope conductance of the ohmic part of the curve was calculated from a linear regression between 40 and +40 mV for each of the 26 independent experiments. The rcoefficients were higher than 0.99. The conductance of the channel is 525 ± 12 pS. B, the substates were observed only at positive potential differences. The I-V relationship of the substate () was fitted by a third-order polynomial regression ( r= 0.985). The I-V curve of the fully open state was redrawn from A ().



The use of different lipid composition such as POPE, POPE/palmitoyloleoyl phosphatidylcholine (1:1, w/w), diphytanoyl phosphatidylcholine, or azolectin has no effect on both frequency of reconstitution and conductance of the channel. The electrical properties of the 525-pS channel measured in the presence of divalent cation in the cis compartment (5 mM Caor 5 mM Mg) were not affected by the presence or the absence of 10 mM EGTA.

The ratio of the permeability coefficient of chloride ions to that of potassium ions is calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The reversal potentials measured in three different cis/trans gradients of 450/150 mM KCl, 150/400 mM KCl, and 1000/150 mM KCl are +5.6 ± 1.6 mV ( n = 4), 3.9 mV ( n = 2), and +8.5 mV ( n = 1), respectively (Fig. 3). The selectivity of Clover Kin these gradients is 1.6 ± 0.2 ( n = 7).


Figure 3: The selectivity of the channel. The selectivity was calculated from the reverse potential measured in three salt gradients buffered with 10 mM Hepes-KOH (pH 7.2). The ionic concentrations in the cis/trans compartments were 450/150 mM KCl (), 150/400 mM KCl (), and 1000/150 mM KCl (). Data were fitted by a third-order polynomial regression (, r = 0.998; , r= 0.999; , r= 0.993).



Effects of the Salt Concentration on the Channel Conductance

Single-channel conductance is measured in symmetrical KCl concentrations ranging from 50 mM to 1 M. The I-V curves measured in salt concentrations of 1 M, 750 mM, and 500 mM KCl can be fitted by a linear relationship (Fig. 4 A). The linear regressions give single-channel conductances of 2.1, 1.7, and 1.2 nS, respectively. At lower salt concentrations (50 and 100 mM KCl), the slope conductances calculated between 40 and +40 mV are 168 and 349 pS, respectively (Fig. 4 B).


Figure 4: Effect of salt concentration on the single-channel conductance. A, the I-V curve was measured in a symmetrical 150 mM KCl, and then KCl concentration was increased to the required concentration by the addition of aliquots of concentrated KCl to each compartment. Each curve represents data points from an independent experiment. The slope conductance was calculated by linear regression. The rvalues were higher than 0.995. Single-channel conductances are 2.1 nS in 1 M KCl (), 1.7 nS in 750 mM KCl (), and 1.2 nS in 500 mM KCl (). represents the current measured in symmetrical 150 mM KCl and redrawn from Fig. 2 A. B, the channel was reconstituted in a salt gradient of 50 mM KCl, 10 mM Hepes-KOH (pH 7.2) in the cis compartment and 10 mM Hepes-KOH (pH 7.2) in the trans compartment. Then, the KCl concentration was increased to make a symmetrical final concentration of either 50 or 100 mM KCl. Each curve represents data points from an independent experiment. The slope conductances were calculated by a linear regression from 40 to +40 mV ( r> 0.990). The conductances were 349 pS in 100 mM KCl ( and 168 pS in 50 mM KCl (). represents the current measured in symmetrical 150 mM KCl from Fig. 2 A. C, the slope conductance is plotted as a function of the conductivity of the experimental solution. Data were fitted with a linear regression ( r= 0.998).



Large channels called porin form water-filled pores across the membrane through which small solutes can diffuse (19, 20, 21, 22) . Their single-channel conductance increases linearly with the solute concentration. This characteristic is also shared by the 525-pS channel whose single-channel conductance is found to be a linear function of the conductivity of the aqueous solution (Fig. 4 C).

Voltage-dependent Gating of the Channel

The study of the voltage dependence is performed in symmetrical 150 mM KCl, 10 mM Hepes-KOH (pH 7.2). Fig. 5A illustrates the open probability of the fully open state at various voltages compiled from 13 bilayers. The channel exhibits an open probability of about 0.2 at voltages ranging from +15 to +60 mV. The slight decrease of the open probability observed after +60 mV is not statistically significant. At negative potentials, the open probability decreases to zero. The open probability of the substate is shown in Fig. 5 B. The substate becomes a long-lived state after +40 mV. The open probabilities of the substate calculated at +40 and +60 mV are statistically different.


Figure 5: Voltage dependence of the channel. The experiments were performed in symmetrical 150 mM KCl, 10 mM Hepes-KOH (pH 7.2). Single-channel open probabilities were estimated using digitized data of either 30- or 60-s durations. A, the open probability of being in the fully open state measured at various voltages was compiled from 13 bilayers. B, the open probability of being in the substate measured at various voltages was obtained from 10 bilayers.



Chemical Modification of the Channel

To investigate the gating process of the channel, we used succinic anhydride to convert a positive amino group into a negative carboxyl group (18) . The bilayer lipid membrane is formed in a symmetrical 150 mM KCl solution, and succinic anhydride (3.8 mM, final concentration) is added on each side of the membrane. 5 min after addition of succinic anhydride, the channel is in its open state and closes only on rare occasions when the membrane is polarized at 30 mV (Fig. 6 A). When a positive membrane potential difference of +50 mV is applied, the modified channel remains in the open state for few seconds and afterward completely closes (Fig. 6 A). Fig. 6 B summarizes the open probabilities of the fully open state measured before and after succinylation of the channel. At electrical potential differences ranging from 70 to +40 mV, the open probability reaches the maximum ( P= 1). But at electrical potentials higher than +40 mV, the open probability decreases because the channel shifts either to the substate or to the fully closed state. The succinylation does not seem to change the structure of the pore since the conductance is not affected by the chemical treatment. To measure the effect of succinic anhydride on the ion selectivity of the channel, we have reconstituted ion channels in planar bilayers in the presence of a salt gradient of 1000/100 mM KCl (cis/trans). The I-V relationship is recorded before and after addition of succinic anhydride (3.8 mM) to the cis side. The reversal potential shifts from +8.4 mV ( n = 2) to 8.9 mV ( n = 2) when the channel is succinylated (Fig. 7), which indicates that succinylation of the channel changes its selectivity. The anionic channel becomes cationic with a selectivity for Kover Clof 0.67 instead of 1.6 calculated for the unmodified channel.


Figure 6: Effect of succinic anhydride on the open probability of the fully open state. Succinic anhydride (3.8 mM) was added to the cis and trans compartments of a bilayer formed in symmetrical 150 mM KCl and 50 mM MOPS-KOH, pH 7.2. After 5 min of treatment, Pwas determined at various membrane potentials using recordings of either 30- or 60-s durations. Control experiments were performed before addition of the chemical. A, the current traces are typical recordings of the channel activity at 30 and +50 mV. The fully closed state is indicated by c. B, the open probability of the fully open state of the channel treated with succinic anhydride () is compared with that of the unmodified channel (). Data are collected from two independent bilayers () or are the mean calculated from three independent bilayers ().




Figure 7: Effect of succinic anhydride on the ion selectivity. The selectivity of the channel was calculated from the reverse potentials measured in a salt gradient (cis/trans) (1 M KCl, 50 mM MOPS-KOH, pH 7.2, 100 mM KCl, 10 mM Hepes-KOH, pH 7.2). The I-V relationships before () and after () addition of 3.8 mM succinic anhydride were measured for two independent bilayers. Data were fitted by a linear regression ( r= 0.994 and 0.991, respectively).



Dextran sulfate is a large impermeant polyvalent anion (8 kDa) known to decrease the open probability of the mitochondrial VDAC at a concentration of 6 µM(23) . To test if dextran sulfate is a potent inhibitor of the 525-pS channel, it was added on each side of the bilayer (6.3 and 31 µM, final concentrations). Neither the voltage dependence nor the conductance of the 525-pS channel were modified whatever the concentration used (data not shown). Each experiment was repeated four times.


DISCUSSION

We have incorporated a large conductance (525 pS) channel from the inner envelope membrane of the chloroplast in a planar lipid bilayer. To our knowledge, it is the first time that a porin is characterized in the inner envelope of the chloroplast. Like other porin found in the outer membrane of bacteria, chloroplasts, and mitochondria, the 525-pS channel has a large conductance, which increases linearly with salt concentrations (Fig. 4). Moreover, it is weakly anion selective (Fig. 3). Despite these two similarities, the 525-pS porin is different from that found in the outer membrane of chloroplasts and mitochondria. The large conductance channels of the outer envelope membrane of chloroplasts of Nitella are cation selective ( P/ P= 4.6 and 3.5) (3) , and that of spinach chloroplast has a conductance of 7 nS in 1 M KCl, which is one of the largest conductances of all known porins (2) . The probability that the 525-pS channel observed in the inner envelope fraction could be a contamination from the thylakoid membrane or from the outer envelope is very weak. Indeed, the electrical signal characterizing the 525-pS channel was never observed when the outer envelope membrane or the thylakoid membrane of the chloroplast was used instead of the inner envelope fraction.

The VDAC found in the outer membrane of mitochondria also belong to the porin family (20, 21, 22) . Like the 525-pS channel, it is weakly anion selective and voltage dependent. However, our study provides evidence that there are at least five differences between the VDAC and the 525-pS channel. First, the single-channel conductance of 2.1 nS calculated in 1 M KCl is lower than the values reported for mitochondrial VDAC (3.7-4.5 nS). Second, the lifetime of the fully open state of the 525-pS channel is much shorter than that of the VDAC, which is estimated to be at least several seconds (20, 21, 22) . Third, the 525-pS channel exists in three stable conformations: a fully open state, a fully closed state, and a substate. Thus, the 525-pS channel closes to a nonconducting state, whereas the mitochondrial VDAC almost never closes completely and remains either in the fully open state or in the substate. Fourth, the polyanion dextran sulfate (6.3 and 31 µM) has no effect on the conductance and the gating of the 525-pS channel. In contrast, electrostatic interactions between dextran sulfate and the positive gating charges of the mitochondrial VDAC decrease its open probability (23) . Finally, the voltage gating of the 525-pS channel depends on the polarity (Fig. 5); the open probability of the fully open state is higher at positive voltages than at negative voltages, and the substate appears only at positive potentials whereas the open probability of the mitochondrial VDAC is identical whatever the sign of the electrical potential differences and follows a ``bell-shaped'' function.

To investigate the gating process of the 525-pS channel, we performed chemical modifications of the channel with succinic anhydride. Succinylation of proteins is known to convert the positive groups of some amino acids into negative groups (18) . The walls within the pore are probably lined with such positive residues. When we modified the net charge of the pore by converting some of the positive amino acids into negative amino acids, the voltage gating was changed and the open probability of the channel was dramatically increased. However, it did not prevent the channel closure at high positive potentials. These results suggest either that the gating of the chloroplast porin is directly mediated by the positive charges distributed inside the pore or that succinylation of some positive residues just stabilizes the channel conformation in its fully open state.

The conversion of charges also alters the ion selectivity of the channel. When the positive residues lining the pore are converted into negative residues then the local electrostatic force is modified, and the selectivity of the channel is reversed. These results agree with those observed in the bacterial porin PhoE and in the mitochondrial VDAC (19, 24, 25) and suggest that the positive residues form the filter lining of the pore.

The occurrence of a porin channel in the inner envelope membrane of chloroplasts is surprising since such a large channel would dissipate the electrochemical gradient. It has been shown that the inward side of vesicles will, after fusion, face the solution bathing the trans compartment (26) . Since the inner envelope vesicles are predominantly inside-out (27) , the channels will have their stroma surface facing the cis side of the bilayer. Therefore, under physiological conditions, the channel should be mainly closed because the electrical potential of inner envelope membrane was measured to be about 100 mV (28) . It cannot be ruled out that the 525-pS channel might remain closed by soluble elements present in the chloroplast stroma or inter-envelope space. Large conductance channels involved either in pressure transduction or in import of proteins have been already observed in the inner membrane of mitochondria and bacteria (29, 30, 31, 32, 33, 34) . The reconstitutions of contact sites from brain mitochondria and Escherichia coli have shown that these membrane fractions contain several ion channels with a conductance ranging from 475 pS to 1 nS in 150 mM KCl (35) and from 300 pS to 1.5 nS in 100 mM KCl (30) . The exact mechanism of protein transport across the envelope membranes of chloroplasts is still unknown. It has been suggested that protein translocation proceeds via two distinct protein-conducting channels in the outer and in the inner envelope membranes (36) . Therefore, an alternative explanation could be that the 525-pS channel is a component of the protein import machinery present in the inner envelope of chloroplasts.


FOOTNOTES

*
This work was supported by The Belgian Fund for Scientific Research (FNRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research Assistant of the National Fund for Scientific Research (Belgium).

Research Associate of the National Fund for Scientific Research (Belgium). To whom correspondence should be addressed. Tel.: 32-2-6505383; Fax: 32-2-6505113.

The abbreviations used are: S, siemens; VDAC, voltage-dependent anion channel; MOPS, 3-( N-morpholino)propanesulfonic acid; POPE, palmitoyloleoyl phosphatidylethanolamine; POPS, palmitoyloleoyl phosphatidylserine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We are greatly indebted to Prof. J. M. Ruysschaert for critically reading the manuscript.


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