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J. Biol. Chem., Vol. 281, Issue 25, 17347-17358, June 23, 2006
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From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, January 30, 2006 , and in revised form, April 5, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The voltage-dependent anion-selective channel (VDAC)/mitochondrial porin is the most abundant protein of the OMM. When reconstituted in artificial membrane systems, such as liposomes and planar bilayer membranes (black lipid membrane (BLM)), the VDAC commonly appears as a large channel. At low potentials (<30 mV), the VDAC was found to be mostly fully open (
4.5-nanosiemens conductance in 1 M KCl) and weakly anion-selective, whereas at high potentials, the VDAC showed a switch to cationic selectivity and closed to approximately half of the original conductance (classical "closed" states) (10-13). Since the molecular cut-off of the VDAC is >3,000 Da in the fully open state, the common belief was that the OMM is freely permeable to small molecules and ions. However, during the past decade, several lines of evidence raised questions about the large size pore-forming activity of the VDAC in natural membranes and the consequent high permeability of the OMM (14). Patch clamp measurements of OMM fractions and intracellular patch clamping of native mitochondria did not show high conductance channel activity (15, 16) unless ionic strength or [Ca2+] was elevated to supraphysiological levels (17). Within a cell, the OMM may form a barrier against the transport of some small molecules (e.g. ADP (18, 19)). Furthermore, in certain apoptosis models, the OMM permeability has been shown to be restricted for metabolites (20). Recently, selective permeabilization of the OMM by tBid, a proapoptotic protein (21), and by overexpression of VDAC (22) was reported to enhance the inositol 1,4,5-trisphosphate-induced mitochondrial matrix [Ca2+] ([Ca2+]m) signal. These results suggested that the OMM Ca2+ permeability could limit rapid Ca2+ uptake to the mitochondria during physiological calcium signals. Furthermore, using targeted enhanced yellow fluorescence protein, lower pH was recorded in the mitochondrial intermembrane space (IMS) than that in the cytosol (23). A possible explanation for this phenomenon is that the VDAC did not permit rapid equilibration of the protons across the OMM.
To understand the reason for the apparent discrepancy between reconstituted and nonreconstituted data, we focused our interest on the experimental parameters of the reconstituted systems. We recognized that in the liposome and BLM experiments, typically supraphysiological [Ca2+] were used (0.1-5 mM [Ca2+] in BLM experiments and unbuffered or poorly buffered calcium in liposome experiments), presumably to increase membrane stability. Also, the bilayers were regularly bathed in a 1 M salt solution (e.g. KCl). To establish the VDAC behavior in the presence of physiological (submicromolar) and elevated [Ca2+] as well, we set up both liposomal transport and BLM experiments with purified VDAC and OMM and fluorescence imaging of mitochondrial ion transport in single cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of Dye Release and Ca2+ Uptake by Plain and VDAC-reconstituted Liposomes—Soy lecithin (L-
-phosphatidylcholine (Soy 20%) from Avanti Polar Lipids or 1,2-diacyl-sn-glycero-3-phosphocholine from Sigma) was washed with 10 mM EGTA to remove Ca2+ contamination and was recrystallized. Liposomes were prepared by sonicating 50 mg/ml recrystallized lipid in a buffer containing 100 mM KCl, 20 mM Hepes/KOH, pH 7.2, 325 µM EGTA, and 5 mg/ml dextran. After sonication, incorporation of purified VDAC and loading with fura2FA (50 µM) or with 10-kDa fura-dextran (250 µg/ml) was carried out by subjecting these vesicles to three freeze-thaw cycles. The liposomes were washed three times and were used promptly after the last washing step.
Dye release experiments were carried out essentially as described previously (25). Briefly, 30-µl aliquots of the plain and VDAC-reconstituted liposomes were added to 720-µl aliquots of 100 mM KCl, 20 mM Hepes/KOH, pH 7.2, and EGTA 325 µM supplemented with various amounts of CaCl2 to set different [Ca2+]. After incubation for 0, 5, or 10 min at room temperature, the liposome suspensions were centrifuged at 35,000 x g for 10 min at 4 °C. Subsequently, the fura fluorescence released to the supernatant and the residual liposomal fraction in the solubilized pellet (Triton 0.3%) was measured (excitation, 340 nm; emission, 500 nm). The concentration of CaCl2 was increased to 2 mM in every sample before the fluorescence measurement.
For the Ca2+ transport measurements, the fura-dextran-loaded, VDAC-reconstituted, or plain liposomes were resuspended in a fluorometer cuvette in a buffer containing 100 mM KCl, 20 mM Hepes/KOH, pH 7.2, and 325 µM EGTA at room temperature. To attain various [Ca2+] levels, CaCl2 (100, 200, 300, 400, or 500 µM) was added. The time course of the fura fluorescence was monitored in a PTI DeltaRam fluorometer (excitation, 340 and 380 nm; emission, 500 nm). At the end of the measurement, to permeabilize every liposome for Ca2+, ionomycin (5 µM), a Ca2+ ionophore, was applied. Calibration of the fura ratio in terms of nM [Ca2+] was added carried out as described previously (26).
For calculation of the initial rate of the VDAC-mediated Ca2+ entry, first a curve was fitted to the time course of the intraliposomal [Ca2+]in each condition. The nonreconstituted background displayed a quasilinear kinetics, whereas the VDAC-reconstituted samples gave an excellent fit with the combination of an exponential and a linear component (y = y0 + a x (1 - e-b(x - c)) + d x (x - c)). The derivation of the exponential tag on x = c gives the initial rate that is equal to a x b.
Planar Lipid Bilayer Experiments—Purified asolectin (freshly recrystallized from stock purchased from Sigma or Avanti Polar) bilayers were prepared by painting a decane solution across a hole of a delrin cup (Warner Instruments). The experimental medium contained 0.1 M KCl or 1 M KCl plus 20 mM Hepes, pH 7.2. The preparation and storage of the medium was carefully controlled to minimize the risk of Ca2+ contamination. Under these conditions, the [Ca2+] was 500-600 nM. In order to exclude Ca2+ contamination, at the end of several experiments, the content of the cup was transferred to a fluorometer cuvette, and the [Ca2+] was evaluated by fura2 (0.25 µM) fluorescence measurement. The bilayer was painted, applying a drop of lipid to the hole by the means of a Teflon rod. After a stable membrane appeared (around 80 picofarads) and was recorded for 5 min, isolated VDAC or OMM vesicles were added to the cis partition. The additions of 0.5-6 µl of solubilized VDAC protein (VDAC-prep) or OMM vesicles (OMM-prep) were made in the vicinity of the membrane. The content of the partitions was stirred by magnetic bars. Usually, the channel activity appeared within 10 min. If not, the addition of VDAC-prep was repeated. The total amount of added protein was 0.3-25 ng. In a few cases when the VDAC-prep was added only once, single channel activity appeared. The experiments were carried out at room temperature (22-24 °C).
The cis and trans sides were coupled to electrodes via agar bridges. The electrodes were connected to CV203B headstage (Axon Instruments). For data acquisition, an Axopatch 200B (Axon Instruments) amplifier was used. The output signal of the amplifier was digitized by a Digidata 1200 digitizer. The headstage and chamber were placed in a small aluminum box (Faraday cage) that was closed during the measurements. The box was grounded to the headstage, as is recommended by the Axon Instruments. Noise level was monitored at the readout of the patch amplifier. The activity was monitored and recorded by means of an IBM-compatible PC. The amplifier was used in patch mode. A data point was obtained in every 60 µs (the high rate was needed to detect the flickering bursts). Current recordings were filtered by a built-in low pass, 4-pole Bessel filter of the amplifier (2 or 5 kHz). In some cases, the signal was also digitally filtered (described in legend to Fig. 4). The signal was also collected essentially unfiltered for data analysis. When an ion channel appeared, first, it was recorded for 10-30 s at various voltages, and then it was recorded for at least 5 min at -20 or +20 mV. CaCl2 (100 µM) and EGTA/Tris (200 or 400 µM, pH 7.4) were added to both sides of the membrane. After stirring, the recording was continued for at least 5 min. For the online recording and offline analysis of traces, the PCLAMP 9.0 program package (Axon Instruments) was used.
Imaging Experiments—RBL-2H3 mucosal mast cells were cultured and plated for experiments as previously described (27). Cells (4.5 x 106 in 250 µl) were transfected with plasmid DNA in suspension using electroporation (cytochrome c-GFP (cyto c-GFP) (28) and mitochondrial matrix-targeted DS Red (mito-DS Red); Clontech), keeping total DNA at 30 µg, and then were plated to coverslips. Imaging was carried out 24-36 h after transfection. For imaging of [Ca2+]m, the cells were loaded with fura2FF/AM (5 µM) for 50-60 min at 37 °C in extracellular medium composed of 121 mM NaCl, 5 mM NaHCO3,10mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 2% (w/v) bovine serum albumin, pH 7.4, in the presence of 0.003% pluronic acid. After dye-loading, the cells were rinsed with Ca2+-free extracellular buffer containing 100 µM EGTA. Permeabilization was carried out in 1 ml of ICM (120 mM KCl, 10 mM NaCl, 1 mM KH2PO4,20mM Tris-HEPES, and 1 µg/ml each antipain, leupeptin, and pepstatin at pH 7.2) supplemented with saponin (30-50 µg/ml) and 20 µM EGTA/Tris in the incubation chamber for 5 min (35 °C). Subsequently, cells were put to fresh ICM containing 10 µM EGTA/Tris. Confocal imaging of cyto c-GFP and mito-DS Red was conducted using a Bio-Rad Radiance system, whereas fluorescence imaging of pHIMS and [Ca2+]m was carried out using an excitation wavelength switch (DG4; Sutter) and a high quantum efficiency cooled CCD camera (Pluto; Pixelvision) fitted to an Olympus IX70 microscope. At confocal imaging, cyto c-GFP and mito-DS Red were excited using the 488- and 568-nm lines of a krypton/argon ion laser source, respectively, recording 512 x 512-pixel image pairs at 0.3 s-1. At the fluorescence imaging experiments, cyto c-GFP was excited at 480 nm, and fura2FF was excited at 340 and 380 nm, and images or image pairs were recorded at 1 s-1. Image analysis was performed using custom-made software (26). Fluorescence (averaged from pixels covering whole cell areas) was determined separately for each single cell (35-50 cells/field) on the field.
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1.5 mM. Thus, initially, the [Ca2+] was maintained slightly lower than the physiological [Ca2+]c (<100 nM), and the increase in [Ca2+] included the range of both the global [Ca2+]c signals (<2 µM) and the peak [Ca2+]c attained only in the vicinity of open Ca2+ release or entry channels (up to several hundred µM). For the sake of membrane stability, the bilayers were formed and recorded at 600 nM [Ca2+] (Figs. 4B, 5, 6), and to study the effect of Ca2+, the [Ca2+] was increased to 50 or 100 µM. [Ca2+] in the buffers was calculated using WINMAX software (Stanford University, CA) and was determined regularly by fluorometric [Ca2+] measurements. Experiments were carried out with 3-6 different membrane or cell preparations, and the data are shown as mean ± S.E. Significance of differences from the relevant controls was calculated by Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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32 kDa in the VDAC-prep, whereas among several bands, the dominant one was also the 32-kDa band in the OMM-prep (Fig. 1A, top). The enrichment of the 32-kDa band in the OMM-prep was apparent when compared with the mitochondrial homogenate (Mito), IMM, and CP fractions (Fig. 1A, top). Immunostaining with an anti-VDAC antibody showed intense reaction with the 32-kDa band in the VDAC-prep and also in the submitochondrial membrane fractions (Fig. 1A, middle). The OMM-prep displayed robust immunoreactivity relative to the mitochondrial homogenate, IMM, or CP fractions (Fig. 1A, middle). Several Bcl-2 family proteins reside in the mitochondrial membranes to control the OMM permeabilization. Bcl-xL, an antiapopototic Bcl-2 family protein, was present in the homogenate but was not detectable in the VDAC-prep (Fig. 1A, bottom). By contrast, the OMM-prep was enriched in Bcl-xL (Fig. 1A, bottom). Similar to the Bcl-xL, Bcl-2 and Bax were also undetectable in the VDAC-prep and abundant in the OMM-prep (data not shown). The activity of the monoaminooxidase (MAO), which is frequently used as an OMM marker enzyme, showed a 15-20-fold enrichment in the OMM-prep (Fig. 1B, gray columns), whereas succinate dehydrogenase (SDH), an IMM enzyme, was present in the IMM fraction but was hardly detectable in the OMM-prep (Fig. 1B, open columns). Collectively, these data suggest that the only major protein in the VDAC-prep is VDAC and that the OMM-prep is highly enriched in VDAC. The OMM-prep has little contamination with IMM components but contains OMM proteins, including several Bcl-2 family proteins.
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Ca2+ Dependence of the Permeability of Liposomes Reconstituted with VDAC—To determine whether Ca2+ exerts any effects on the VDAC-mediated transport of small molecules, the fura2 release from plain and VDAC-prep-reconstituted liposomes (VDAC-liposomes) was evaluated at varying Ca2+ concentrations (Fig. 2). The liposomes were exposed to <20 nM, 2-3 µM, and >1mM [Ca2+], respectively, and were incubated for 0 or 5 min at room temperature. Measurement of the liberated and the residual liposomal fluorescence provided information about the rate of fura2 release in the various conditions. No significant effect of Ca2+ was observed on the release of fura2 from the plain liposomes (Fig. 2, left). At subphysiological [Ca2+] (<20 nM), no VDAC-mediated fura2 release occurred, but a dose-dependent release was observed at the higher Ca2+ concentrations tested (Fig. 2, right). Importantly, the VDAC-liposomes were extensively washed prior to exposure to Ca2+, eliminating the possibility that the effect of Ca2+ was mediated by insertion of surplus VDAC. The low permeability of the VDAC-liposomes for fura2 indicates that at low [Ca2+] and physiological [K+], the VDAC might form a barrier against small molecules. Furthermore, the permeation of small molecules through the VDAC seems to increase when [Ca2+] is increased to the micromolar range.
To test the effect of [Ca2+] on the Ca2+ permeability of VDAC, liposomes were loaded with a fura-conjugated dextran (10 kDa), which did not exit the VDAC-liposomes even at 100 µM [Ca2+] (Fig. 1C). The Ca2+ influx was monitored at varying extravesicular [Ca2+] (Fig. 3). Ca2+ influx was initiated by the addition of Ca2+. The kinetics of the Ca2+ influx for plain and VDAC-reconstituted liposomes at two different concentrations is shown in Fig. 3, A and B. The addition of 200 and 500 µM CaCl2 increased the [Ca2+] to 500 nM and 4 µM, respectively. The fura excitation ratio showed a rapid and dose-dependent rise in luminal [Ca2+] in the VDAC-reconstituted liposomes, indicating Ca2+ permeation through the VDAC. To further characterize the [Ca2+] dependence of the VDAC-mediated Ca2+ entry, the initial rate was calculated at each extraluminal [Ca2+] (for a description, see "Materials and Methods"). The luminal [Ca2+] in the nonreconstituted liposomes displayed a quasilinear rise, whereas in the VDAC-liposomes, it was described by the sum of a linear and an exponential component. Thus, the exponential component seems to describe the VDAC-dependent Ca2+ influx into the reconstituted population of liposomes (the yield of reconstitution was 20-30%). In Fig. 3C, the calculated initial rates were plotted against extraluminal [Ca2+] for three separate experiments (closed circles, triangles, and open circles). In each experiment, a steep, supralinear rise in the initial rate was observed in the 2-3 µM range of extraluminal [Ca2+], indicating an increase in the permeability to Ca2+. Thus, these data suggest that the Ca2+ permeability of VDAC is regulated by cytoplasmic [Ca2+]. Both the dye release and the Ca2+ influx experiments are in support of the regulation of VDAC by [Ca2+] 600 nM.
Isolated VDAC Forms Small Channels in Low Calcium Medium—Previous BLM studies of the VDAC were commonly carried out in 0.5-1 M KCl, and 
100 µM CaCl2 was typically present. To evaluate the possible role of the physiological ion concentrations in the gating of VDAC, we carried out BLM experiments in 100 mM KCl (symmetrical), both in the presence and absence of 100 µM CaCl2. When the VDAC-prep was fused to planar bilayers in the presence of 100 µM CaCl2 (Fig. 4A), it showed the classical hallmarks of the VDAC, such as the characteristic size (single channel full conductance of about 450 pS in 100 mM KCl), symmetrical voltage dependence (practically permanent, full open state at lower voltages (+20 mV, top) and closures on higher voltages (-50 mV, middle)), a plethora of subconductance states, and slow gating kinetic (bottom). Thus, these results have reproduced the core findings of previous BLM studies of isolated VDAC.
In the absence of CaCl2 (in a nominally Ca2+-free buffer, [Ca2+] 600 nM), we recorded channels in 48 experiments. In 43 cases, only small conductances were found (Fig. 4B). In four cases, no channel activity appeared, but the addition of 100 µM CaCl2 induced gating. In one case, we observed channel activity similar to the activity found in the presence of CaCl2 (not shown). However, it remains a possibility that the chamber was contaminated with CaCl2 in this particular case. In Fig. 4B, a short multichannel recording of VDAC activity at 600 nM [Ca2+] is presented. At 20 mV, a nearly closed substate (30 pS) was the dominant conductance that was interrupted by short opening events. The amplitudes were typically smaller than the half-size of the full conductance (see histogram). Occasionally, amplitudes above the full conductance of a single channel (480 pS) also appeared, indicating the presence of more than one channel in the membrane (not shown). This activity is in sharp contrast to the permanent full conductance opening that was observed in the high calcium condition at 20 mV (Fig. 4A). It is of significance that the most frequent subconductance states observed in submicromolar [Ca2+] medium are smaller in size than the "classical closed states" that were established at high voltage (>30mV)in1 M KCl and high [Ca2+].
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Calcium Induces Increased VDAC Activity and Transition to Full Opening—The experiments shown in Fig. 4 have revealed the strong impact of steady 100 µM (A) and submicromolar (B and C) [Ca2+] on VDAC activity. We investigated whether the addition of calcium induces transition between the low conductance and the classical high conductance state. In 19 experiments, after the channel activity was recorded at 600 nM [Ca2+], the [Ca2+] was raised to 50 or 100 µM. In 16 experiments, the channel activity was clearly affected by the Ca2+ addition (in four cases, gating appeared only after the Ca2+ addition), whereas in three experiments, there was virtually no change. Two types of responses to Ca2+ were seen: an immediate increase in activity and, after a lag period, an eventual transition to full opening. To illustrate both effects of Ca2+, in the upper row of Fig. 5A, the complete traces recorded at 600 nM [Ca2+] (i) and after elevation of [Ca2+] (ii) in a multichannel experiment are shown. In the first 15 s of [Ca2+] elevation, the activity was augmented, and at 16 and 115 s, the conductance was largely increased. The size of the increase at 16 s is consistent with the opening of one VDAC to the full conductance state, and the increase at 115 s indicates the opening of a second channel. At the top of the second opening, additional activity appeared, which demonstrated the presence of at least one more VDAC. To better visualize the initial effect of Ca2+ on the channel activity, a short segment of the records obtained both at 600 nM [Ca2+] and 10 s of the [Ca2+] elevation is also shown on an expanded time scale (iv and v). The individual events seem to be shorter and attain larger conductances after the [Ca2+] rise. The average current was increased from 0.86 to 1.51 pA in the initial period of the Ca2+ effect and to 10.62 pA after the opening of the first channel to full conductance (Fig. 5A, vii). All-point amplitude histograms revealed a Ca2+-induced shift in the amplitude spectrum from low to higher values (iii and vi). The histogram of the whole trace displays two additional peaks corresponding to the first and second full openings (10 and 20 pA, respectively; iii). Amplitude analysis discriminated five different subconductance levels in the control trace and two more after Ca2+ addition. The open probability of the first level (0.63 pA, 31 pS) decreased from 0.85 to 0.22, and the second (1.11 pA, 55 pS) and third (1.82 pA, 91 pS) increased from 0.12 to 0.37 and from 0.01 to 0.31, respectively. The two Ca2+-dependent conductances were 4.42 and 9.47 pA (220 and 470 pS, respectively). The latter corresponds to the full conductance. However, the related open probabilities are almost negligible (0.01 and 0.02). Collectively, these results indicate that the channel became faster and individual events became shorter (v), and the average current increase is due to the more frequent occupancy of higher subconductance levels. However, the more frequent gating (and sporadic visits to full conductance) reflects instability of the channel that is later stabilized as classical VDAC with a full open state at low voltage. Along the line of the models that consider the VDAC as the OMM component of the permeability transition pore (PTP), this transition may reflect the VDAC opening when the VDAC participates in the Ca2+-induced permeability transition.
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6 min, we added EGTA to lower the [Ca2+] back into the submicromolar range. One example is shown in Fig. 5B. Ideal histograms of the plot were obtained from the event list of about 6 min long in the control (black), Ca2+-exposed (red), and EGTA-buffered periods (green). The histograms resolved six subconductances. The Ca2+-induced frequency increase is reflected in the elevation of the number of the events of the third, fourth, and fifth subconductance levels (red). Restoration of the submicromolar [Ca2+] by EGTA led to a return to the pre-Ca2+ subconductance pattern (green). This result demonstrates that the Ca2+-induced activation of the VDAC was fully reversible. Taken together, the electrophysiology data complement and extend the results of the liposome experiments. The liposome experiments showed that at submicromolar [Ca2+], the VDAC exerts a restricted permeability to Ca2+ and a relatively small molecular cut-off size. These results were unexpected, based on previous observations of a large pore size of the VDAC in high [Ca2+] environment. The BLM experiments revealed, however, that under low [Ca2+] conditions, the VDAC gates almost exclusively with small subconductances. In addition, the Ca2+-induced reversible increase in channel activity, more specifically the higher conductance levels, seems to provide a mechanism for the Ca2+-dependent permeability increase in the VDAC-liposomes. The next question is whether the small conductance gating and the calcium sensitivity of the isolated VDAC are physiologically relevant phenomena. As an approach to this question, we investigated the ion channel activity in the natural outer mitochondrial membrane (OMM-prep).
Calcium Controls VDAC Activity in Natural Mitochondrial Outer Membrane—First, noncontact OMMs were fused into BLM membranes and were exposed to 1 M KCl and 100 µM CaCl2. The records showed "classical VDAC"-like activity, symmetrical voltage dependence (practically permanent open state at lower voltages and closures at higher voltages), numerous subconductance states, and slow gating kinetics. In Fig. 6A, the upper graph illustrates the voltage-dependent closure in a multichannel experiment (five channels, single channel full conductance of about 4.5 nanosiemens in 1 M KCl). The lower graph with an expanded scale shows several substates. Both traces display slow gating kinetics.
In 100 mM KCl and submicromolar [Ca2+], there were always various small conductance gatings found (30 experiments). The activity appeared in burstlike fashion, which is exemplified in Fig. 6B (see sample on extended time scale). It is also shown in Fig. 6B that the Konig's polyanion, an inhibitor of the VDAC, practically eliminated the OMM channel activity, although some rare events remained, verifying the presence of the channel (10 of 10 experiments). The OMM-prep is highly enriched in VDAC (Fig. 1), and upon fusion to BLM, it provided classical VDAC activity in 1 M KCl and appeared as a polyanion-sensitive channel, suggesting that the OMM-derived channel represents VDAC activity in the natural membrane. Along this line, the small conductance gating in 100 mM KCl seems to apply to the VDAC in the natural membrane at the physiological ion concentrations.
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[Ca2+]c Controls the H+ and Ca2+ Permeability of the OMM in Cells—To validate the biophysical data in a cellular paradigm, we sought for an approach that allows detection of materials entering mitochondria through the VDAC directly in the IMS. EGFP and its variants have been used for pH measurements (29), and EGFP has been targeted to the IMS as a cytochrome c fusion protein (28). We set out to generate a pH gradient between cytosol and IMS and to record the protons that traverse the OMM in the IMS using cyto c-GFP (Fig. 7A, i).
Confocal images show the distribution of both cyto c-GFP (green) and mito-DS Red (red) in a permeabilized RBL-2H3 cell in Fig. 7A, ii. Cyto c-GFP appeared in the structures positive for the red mitochondrial marker. In response to tBid, a proapoptotic Bcl-2 family protein that selectively permeabilizes the OMM, complete loss of cyto c-GFP occurred, whereas the mito-DS Red fluorescence remained unchanged (Fig 7A, ii, graph and images on the right). Thus, cyto c-GFP is confined to the IMS in the transfected RBL-2H3 cells.
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To explore whether ATP permeation across the OMM was also enhanced by Ca2+ in the above described model, the ATP-driven Ca2+ accumulation was monitored by imaging [Ca2+]m (Fig 7B, i). Specifically, RBL-2H3 cells were loaded with fura2FF using a protocol that results in compartmentalization of the dye in the mitochondrial matrix (26, 27). The fura2FF-loaded cells were permeabilized in the buffer used in the cyto c-GFP imaging experiments, except that oligomycin was omitted. Under this condition, no driving force for the mitochondrial Ca2+ uptake remained. Consistently, Ca2+ ([Ca2+]c = 4 µM) failed to induce any considerable [Ca2+]m elevation (Fig. 7B, ii, red). However, the addition of ATP to the Ca2+-pretreated cells evoked a rapid and large [Ca2+]m increase (Fig. 7B, ii, red). By contrast, when Ca2+ was added at the same time as ATP, only a slow [Ca2+]m rise appeared (Fig 7B, ii, black trace and bar charts). Thus, elevation of [Ca2+]c before ATP addition led to an increase in the rate of the ATP-driven mitochondrial Ca2+ accumulation. VDAC inhibitors could not be used to confirm the involvement of the VDAC in this process, since DIDS and the Konig's polyanion caused loss of the 
m and, at higher concentration, cytochrome c release (25). However, earlier studies have established that the VDAC serves as the primary transport mechanism for both nucleotides and Ca2+ across the OMM (30, 31). Although the studied process involved the transport of ATP and Ca2+ across both the OMM and IMM, the function of the adenine nucleotide translocase is affected by [Ca2+]c only in the millimolar range (32), and the uniporter also shows a relatively modest sensitization at the tested [Ca2+]c (33). Considering these points, the massive increase in the rate of [Ca2+]m rise indicates the possible enhancement of the VDAC-mediated passage of ATP and/or Ca2+ across the OMM by Ca2+. This result complements the observation that Ca2+ stimulates the proton transport across the OMM. Furthermore, both the Ca2+-dependent stimulation of the proton and the ATP/Ca2+ entry serve as evidence that the observations made in the liposome and bilayer experiments are relevant for mitochondrial membrane transport in the cell.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results provide a clue to the apparent discrepancy between the classical electrophysiology work that established the large conductance and almost continuous opening of the VDAC at low voltages (10, 13, 34) and some recent studies of the OMM in the intracellular milieu, which showed the presence of small conductances and restricted permeability to ions or metabolites (17, 18, 21, 22). The classical electrophysiology on isolated VDAC was carried out in the presence of high CaCl2 (100 µM-1 mM) or unbuffered [Ca2+], whereas in the studies of VDAC in the intracellular milieu, the channel was exposed to 100 nM cytoplasmic [Ca2+]. In light of our data on the Ca2+ sensitivity of the VDAC activity, the difference in [Ca2+] can be a source of the above differences in VDAC activity. However, in addition to the different Ca2+ levels, some other factors are also important for the divergent properties of the VDAC in previous works. In the present study, permanent large conductance opening of the isolated VDAC could be triggered by high [Ca2+] in 100 mM KCl, whereas a similar response in the noncontact OMM could be evoked only in the presence of 1 M KCl. The OMM-prep contains VDAC and several other proteins that interact with VDAC. Both a soluble mitochondrial protein and hexokinase-1 have been shown to decrease the VDAC conductance (35, 36), whereas Bax was reported to increase the pore size (37, 38). Thus, the high salt concentration could be required to disrupt a protein-protein interaction that modulates the VDAC activity in the OMM. Importantly, we used noncontact OMM, and the protein-protein interactions of the VDAC are different in the noncontact and contact site regions of the OMM. As to the VDAC at the contact sites, evidence has been presented that the VDAC is a component of the PTP complex (39), and the sustained full conductance opening of the VDAC has been linked to the opening of the PTP (40, 41).
Based on the above data, we predict that at the resting level of cytoplasmic [Ca2+], the VDAC activity and the OMM permeability for Ca2+ and small molecules is regulated. The role of VDAC in the transport of substrates through the OMM was underscored by the inhibition of mitochondrial metabolic function in the presence of agents that induce the classical "closed" states (35, 42). Evidence has also been provided for Ca2+ permeation through the VDAC (31). However, in the cell, the OMM Ca2+ permeability seems to limit the calcium signal propagation to the mitochondria (21), and this restriction can be alleviated by overexpression of the VDAC (22). Importantly, mitochondrial Ca2+ uptake is often driven by [Ca2+] spikes and oscillations that consist of short lasting events and require instantaneous activation of the Ca2+ transport. The relatively slow release of fura2 from VDAC liposomes at low Ca2+ also indicates that small molecules, like substrates, do not necessarily freely traverse the OMM. Notably, restriction of the ADP diffusion through the OMM has been documented (18). However, the transport of substrates is driven by relatively sustained gradients, and the organization of metabolism into complexes may also support channeling of metabolites. One might also speculate that a slow release of protons through the OMM may result in more effective chemiosmosis (43). The Ca2+-induced enhancement of the OMM permeability supports rapid Ca2+ delivery to the uniporter, the channel that mediates Ca2+ uptake via the IMM (44). Through this mechanism, Ca2+-dependent activation of the VDAC facilitates mitochondrial Ca2+ uptake and Ca2+ buffering and may also synergize with the Ca2+-dependent activation of the mitochondrial dehydrogenases and other enzymes to increase ATP production.
In the present work, the VDAC activity was studied in the <20 nM to 100 µM range of [Ca2+]. Clearly, the physiological range of bulk cytoplasmic [Ca2+] is confined to the lower end of this [Ca2+] span (from 50 nM to 1-2 µM). Thus, it is of significance that we documented Ca2+-induced changes in VDAC permeability for Ca2+ and fura2 in this narrower range. Furthermore, it has been demonstrated that a fraction of the mitochondria is localized in the vicinity of endoplasmic reticulum Ca2+ release channels and may be exposed to substantially higher local [Ca2+] than the bulk [Ca2+] elevation (27, 45, 46). Strikingly, elementary Ca2+ release events, known as Ca2+ sparks, have been found to suffice to activate the low affinity Ca2+ uptake in adjacent mitochondria (47). Based on the activation of mitochondrial Ca2+ uptake, we have calculated that the OMM may be exposed to at least 10-20 µM [Ca2+] during the inositol 1,4,5-trisphosphate receptor or ryanodine receptor-linked calcium signal (27). Thus, at least for the mitochondria that are localized close to the sarcoendoplasmic reticulum, the physiological [Ca2+] range extends to the full range used in the present work.
The VDAC belongs to the family of 
-barrel membrane proteins that are mostly confined to the OMM in mammalian cells (48-50). Some hallmarks of the VDAC activity have been firmly established, including the symmetrical voltage dependence, the voltage dependent closure, the presence of numerous substates, slow gating kinetics, and the modulation of activity by the polyanion and DIDS. However, the electrophysiologic analysis of the VDAC has been hindered by the technical challenge posed by the mitochondrial patch clamp recording and also by the slow kinetics of the channel. For many ion channels, 1-s recording provides a sufficient number of events for statistical analysis, whereas the VDAC requires 10-30-min recordings to produce a similar number of events. In the present work, we documented VDAC activity in the entire range, from the completely closed to the fully open state, at physiological Ca2+ and K+ levels. As to the identification of the VDAC, we have reproduced the above described hallmarks of the VDAC for this channel activity and also reproduced the sustained full conductance opening and closure to half-conductance level, when the VDAC-prep was exposed to the conventional recording conditions (high [Ca2+] and [K+]). Thus, under physiological ionic conditions, the VDAC can adopt conformations that appear as a completely closed state or subconductances smaller than the classical "closed" states.
Data collection for relatively long periods allowed us to visualize bursts in VDAC activity, which seem to last for tens of seconds, further illustrating the slow gating of this channel, and to analyze some properties of the events at both submicromolar and 100 µM Ca2+ levels. Our data suggest that Ca2+ serves as an inducer of the large conductances. The Ca2+ sensitivity of the VDAC could be conferred by either a Ca2+-sensitive enzyme that targets the VDAC or by calmodulin that may bind both Ca2+ and the VDAC or by a direct effect of Ca2+ on the VDAC. Since the effect of Ca2+ on the VDAC was observed in a highly purified VDAC-prep and because Ca2+ binding to purified and solubilized VDAC has also been shown (31), a Ca2+ binding site on the VDAC may be primarily considered as the mediator of the Ca2+ effect. Although no EF-hand seems to be present in the sequence of the mitochondrial VDAC, further sequence analysis seems to be warranted, since a motif search with the Gibbs sampler algorithm indicates the presence of conserved sequences that show considerable homology with parts of the EF-hand (not shown). Similar to the natural composition of the OMM, in the present study, cardiolipin was present in the liposomes and bilayers, which may bind Ca2+ and also confer Ca2+ sensitivity to the VDAC. Importantly, the Ca2+-induced shift to large conductances appeared promptly, but the permanent opening to full conductance was typically delayed. A possible mechanism is the dimerization of the VDAC (40, 51). The Ca2+-dependent patterns described here may be modulated in the cell by the generation of electrical potential across the OMM (52); by the pH (53); by the interaction of VDAC with other ions and small molecules, including NADH (54), purine nucleotides (55-57), and superoxide (25); and with a range of proteins that have been listed above. Along the line of a commonly proposed model, the VDAC interacts with the adenine nucleotide translocase and cyclophilin D to form the PTP, which is primarily regulated by [Ca2+]m. This regulation was envisioned to depend on Ca2+ sensing by cyclophilin D, but recent data show that the Ca2+ sensitivity of the permeability transition is not lost in cyclophilin D knock-out mice (58). Thus, Ca2+ sensing by the adenine nucleotide translocase (32) or by the VDAC (although the VDAC is not exposed to [Ca2+]m) may also be of significance for the permeability transition.
The main point of the present work is that the VDAC may display the full range of potential activities in the cell, depending on the Ca2+ concentration. Extension of the previously described permanent large conductance activities to the range of small conductances has major implications for mitochondrial membrane transport. Gating of VDAC by Ca2+ may present a novel mechanism for the calcium signal to control both mitochondrial and cellular functions.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Suite 261 JAH, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-503-1427; Fax: 215-923-2218; E-mail: gyorgy.hajnoczky{at}jefferson.edu.
2 The abbreviations used are: IMM, inner mitochondrial membrane; BLM, black lipid membrane; [Ca2+]c, cytoplasmic [Ca2+]; [Ca2+]m, mitochondrial matrix [Ca2+]; CP, contact point; GFP, green fluorescence protein; IMS, mitochondrial intermembrane space; OMM, outer mitochondrial membrane; OMM-prep, solubilized OMM vesicles; VDAC, voltage-dependent anion-selective channel; VDAC-prep, solubilized VDAC protein; pS, picosiemens; cyto c, cytochrome c; mito-DS Red, mitochondrial matrix-targeted DS Red, DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
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