An Interaction between Bcl-x_L and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca²⁺ Uptake

Background: Ca²⁺ moves across the outer mitochondrial membrane (OMM) through the voltage-dependent anion channel (VDAC).

Results: Disrupting the interaction between VDAC and the antiapoptotic protein Bcl-x_L reduces mitochondrial Ca²⁺ uptake.

Conclusion: Bcl-x_L/VDAC interactions promote Ca²⁺ uptake by increasing transfer across the OMM.

Significance: Mitochondrial matrix Ca²⁺ is tightly regulated at the OMM by the modulation of VDAC.

Abstract

The role of the antiapoptotic protein Bcl-x_L in regulating mitochondrial Ca²⁺ ([Ca²⁺]_mito) handling was examined in wild-type (WT) and Bcl-x_L knock-out (Bcl-x_L-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca²⁺ transients that produced a larger [Ca²⁺]_mito uptake in WT cells compared with Bcl-x_L-KO. In permeabilized cells, stepping external [Ca²⁺] from 0 to 3 μm also produced a larger [Ca²⁺]_mito uptake in WT; moreover, the [Ca²⁺]_mito uptake capacity of Bcl-x_L-KO cells was restored by re-expression of mitochondrially targeted Bcl-x_L. Bcl-x_L enhancement of [Ca²⁺]_mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca²⁺ permeability pathway that directly interacts with Bcl-x_L. Bcl-x_L interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-x_L binding. Peptides reduced [Ca²⁺]_mito uptake in WT but were without effect in Bcl-x_L-KO cells. In addition, peptides reduced [Ca²⁺]_mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-x_L interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-x_L and VDAC promotes matrix Ca²⁺ accumulation by increasing Ca²⁺ transfer across the outer mitochondrial membrane.

Introduction

The Bcl-2 proteins are a family of apoptosis regulators that contain both pro- and antiapoptotic members. Apoptotic stimuli cause activation of proapoptotic members that converge on the mitochondria to increase their membrane permeability. The subsequent release of apoptogenic factors, such as cytochrome c, represents a critical step in the cell death signaling cascade. This process is held in check by the antiapoptotic Bcl-2 proteins, which preserve mitochondrial membrane integrity and inhibit cell death by directly binding and sequestering the proapoptotic members (1).

In addition to influencing mitochondrial permeability directly, Bcl-2 proteins also impinge on apoptotic signaling through their ability to regulate Ca²⁺ homeostasis (2). We and others have shown that this can be mediated through direct interactions between the endoplasmic reticulum (ER)³-localized inositol 1,4,5-trisphosphate receptor (InsP₃R) Ca²⁺ release channel and antiapoptotic Bcl-2 members, including Bcl-2 itself, Bcl-x_L, and Mcl-1 (3–7). The functional consequences of these interactions are complex with both inhibition and stimulation of Ca²⁺ signals being reported. This apparent paradox likely reflects differences among Bcl-2 family members in their binding determinants and abilities to allosterically modulate the InsP₃R (8). Collectively, these data have given rise to a generalized model in which antiapoptotic Bcl-2 proteins confer protection either by limiting Ca²⁺ overload during apoptotic stimuli or by priming cellular resistance through enhanced mitochondrial metabolism (2).

Our previous studies have demonstrated that Bcl-x_L binds to all three InsP₃R isoforms to increase their sensitivity to low levels of InsP₃ stimulation (4–6). This results in increased Ca²⁺ signaling and bioenergetics, a requirement for Bcl-x_L to be maximally effective as an antiapoptotic mediator. More recently, we used mouse embryonic fibroblast (MEF) cell lines derived from Bcl-x_L knock-out (Bcl-x_L-KO) embryos to examine the effect of targeting Bcl-x_L to specific organelles (9). When expressed exclusively at the ER, Bcl-x_L had predicable effects on Ca²⁺ handling but surprisingly did not confer any apoptosis protection. In contrast, targeting to the mitochondria alone was protective; however, the maximal antiapoptotic activity of Bcl-x_L was only seen when it was present at both the ER and mitochondria. One interpretation of these data would be that localization of Bcl-x_L at the mitochondria is required to sense Bcl-x_L-regulated InsP₃R-dependent Ca²⁺ signals.

Uptake of Ca²⁺ into the mitochondrial matrix requires transport across two membranes. Across the inner membrane, this is governed by the calcium uniporter, a channel whose properties have been characterized (10) but whose molecular identity has only recently been described (11, 12). Movement across the outer membrane is mediated largely by the voltage-dependent anion channel (VDAC), a porin channel that serves as the major diffusion pathway for ions and metabolites (13). Of the three VDAC isoforms, VDAC1 is the most abundantly expressed (14) and extensively characterized (13). Indeed, due to the abundance and large pore size of VDAC1, the outer membrane was originally thought to be freely permeable to Ca²⁺, although recent evidence suggests that it may function as a more regulated Ca²⁺ permeability (15–17).

Intriguingly, Bcl-x_L binds directly to VDAC1; however, there is a lack of consensus as to whether it serves to promote (18) or inhibit (19) the function of the channel as a permeability pathway for metabolites and large anions. Similarly, in electrophysiological studies, Bcl-x_L has been reported to both increase (18) and decrease (20) conductance of VDAC1 reconstituted in artificial membranes. It is not known whether Bcl-x_L regulates mitochondrial Ca²⁺ uptake by interacting with VDAC1 or other VDAC isoforms. The purpose of the current study was to define how the interaction between Bcl-x_L and VDAC shapes mitochondrial Ca²⁺ signaling.

EXPERIMENTAL PROCEDURES

Cell Culture

The generation of MEF cells from wild-type and Bcl-x_L-KO embryos and selection of cell lines stably expressing Bcl-x_L targeted to the ER or mitochondria in the KO background have been described previously (9). MEF cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Gemini, West Sacramento, CA), 100 units ml⁻¹ penicillin (Mediatech), and 100 μg ml⁻¹ streptomycin (Mediatech) in an incubator with 95% humidity and 5% CO₂ at 37 °C.

Solutions and Reagents

Hanks' balanced salt solution contained 137.9 mm NaCl, 5.33 mm KCl, 0.44 mm KH₂PO₄, 0.34 mm Na₂HPO₄, 5.56 mm glucose, 4.17 mm NaHCO₃, 1.8 mm CaCl₂, 0.49 mm MgCl₂, 0.41 mm MgSO₄, 10 mm HEPES, pH 7.4 with NaOH. To prepare Ca²⁺-free Hanks' balanced salt solution, CaCl₂ was substituted with MgCl₂, and 1 mm EGTA was added. Intracellular-like medium (ICM) contained 120 mm KCl, 10 mm NaCl, 1 mm KH₂PO₄, 20 mm HEPES, 2 mm sodium succinate, 1 mm EGTA, pH 7.1 with KOH. The free [Ca²⁺] was adjusted to the desired level by varying the ratio of Ca²⁺/HEDTA, calculated using Maxchelator (C. Patton, Stanford University, CA). The following pharmacological reagents were obtained from the indicated sources: digitonin, oligomycin, and FCCP (Sigma-Aldrich); rotenone (MP Biomedicals, Santa Ana, CA); cyclosporin A (Enzo Life Sciences Inc., Farmingdale, NY); dithiothreitol (DTT) (Fisher Scientific); Sarkosyl (IBI Scientific, Peosta, IA); CHAPS (Calbiochem); ruthenium red and valinomycin (EMD Millipore, Billerica, MA); Rhod-2 AM and tetramethylrhodamine (TMRE) (Invitrogen), and Fluo-2 AM (TEFLabs, Inc., Austin, TX). The following peptides based on the human VDAC1 sequence were synthesized by Biomatik (Wilmington, DE): control (LVLGYEGWLA), N-terminal (GLGKSARDVFTKGYGFG), and L14-15 (LAWTAGNSNTR). Cell-permeant versions were tagged with antennapedia homeodomain-derived antennapedia (Antp; RQIKIWFQNRRMKWKK) at the C terminus of each peptide. Anti-VDAC1 mAb was purchased from EMD Millipore.

GST Pulldown Assay and Western Blot

Mouse VDAC1, -2, and -3 cDNAs were subcloned into pGEX-6P-1 (GE Healthcare). Recombinant GST fusion proteins were expressed in Escherichia coli and purified using glutathione-Sepharose beads (GE Healthcare) as described (6). Beads were then incubated with cell lysates or recombinant Bcl-x_L (2 μg) as described (6). Samples were analyzed by Western blot using antibodies against Bcl-x_L (BD Biosciences) and GST (ViroGen Corp., Watertown, MA) with anti-β-actin (Sigma-Aldrich) as a loading control.

Isolation of Mitochondrial and Mitoplast Preparations

Mitochondria were isolated by following established protocols (21). Briefly, MEF cells were harvested and homogenized in mitochondrial isolation buffer (200 mm mannitol, 70 mm sucrose, 1 mm EGTA, 10 mm HEPES, 0.05% BSA, protease inhibitor mixture, pH 7.4 with KOH). Following centrifugation, the mitochondrial fraction was resuspended in ICM buffer prior to experimentation. Mitoplasts were generated by incubating isolated mitochondria in 4 volumes of hypotonic solution (5 mm sucrose, 1 mm EGTA, 5 mm HEPES, pH 7.4 with KOH) for 20 min and then equilibrated on ice with 1 volume of hypertonic solution (750 mm KCl, 80 mm HEPES, 1 mm EGTA, pH 7.4 with KOH) before centrifugation. For Western blot analysis of isolated mitochondria and mitoplasts, samples were lysed and analyzed using standard approaches and blotted with anti-uncoupling protein 3 (Alpha Diagnostic International Inc., San Antonio, TX) and anti-cytochrome c (Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Bcl-x_L Knockdown Mediated by Lentivirally Encoded shRNA

Lentiviral transduction particles (Sigma-Aldrich) carrying Bcl-x_L shRNA or empty vector controls were added to cells in culture, and selection antibiotic (puromycin; 2 μg ml⁻¹) was added after 3 days. Single colonies were then isolated by limited dilution, and Bcl-x_L knockdown was confirmed by Western blot.

Cytoplasmic and Mitochondrial [Ca²⁺] Measurement

For simultaneous measurement of cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) and mitochondrial Ca²⁺ ([Ca²⁺]_mito), cells cultured on glass coverslips were loaded with 3 μm Rhod-2 AM by incubation at 37 °C for 30 min followed by 10 μm Fluo-2 AM at room temperature for a further 40 min. In experiments requiring the flash photolysis of caged Ca²⁺, the membrane-permeable caged EGTA compound o-nitrophenyl EGTA AM (5 μm) was added along with Fluo-2 AM, and Ca²⁺ was photoreleased by brief pulses (150–550 ms) of UV light delivered uniformly throughout the image field. Coverslips were then mounted in a recording chamber positioned on the stage of an inverted microscope (IX71, Olympus America Inc., Center Valley, PA). Cells were visualized using a PlanApo 60×, 1.42 numerical aperture oil immersion objective, and confocal images were acquired using a VT-Infinity 3 (VisiTech International, Sunderland, UK). Fluo-2 and Rhod-2 were alternately excited using the 488- and 568-nm lines, respectively, of a krypton-argon laser. The emitted fluorescence was filtered using a dual bandpass filter set (VisiTech International) and collected and analyzed using HCImage software (Hamamatsu Corp., Sewickley, PA). The chamber was continuously perfused with Hanks' balanced salt solution at room temperature, and a rapid solution changer was used to switch the composition of the solution bathing the cells under study. For isolated mitochondria and mitoplasts, samples were prepared from MEF cells, loaded with Rhod-2, and imaged as described above. Wide field fluorescence microscopy was used to measure [Ca²⁺]_mito in plasma membrane-permeabilized preparations. Rhod-2-loaded cells were mounted in a recording chamber on the stage of an inverted IX71 microscope (Olympus America Inc.) and excited at 548 nm. Emitted fluorescence was filtered at 605 nm and collected using a charge-coupled device-based imaging system running SimplePCI software (Hamamatsu Corp.). The solution perfusion and exchange system was similar to that described above. Cells were permeabilized by 3–4-min exposure to digitonin (25 μg ml⁻¹) applied in Ca²⁺-free ICM. The permeabilized preparation was then allowed to equilibrate in regular Ca²⁺-free ICM for 15 min prior to experimental recording.

Confocal and Electron Microscopy

To image ER and mitochondria confocally, cells were first transfected with eYFP-ER plasmid (Clontech) using an Amaxa Nucleofector device (Lonza, Allendale, NJ) and seeded onto 35-mm glass coverslips. After 16–24 h, the cells were labeled with CellLight® Mitochondria-RFP (Invitrogen) according to the manufacturer's instructions. After a further 24 h in culture, coverslips were mounted in a chamber, and confocal images were acquired using a VT-Infinity 3 system as described above. To image ER and mitochondria using electron microscopy, cells were plated onto ACLAR film disks (Jed Pella, Redding, CA) in a 12-well tissue culture plate with 20,000 cells in each well. After incubation for 24 h, cells were fixed with 3% glutaraldehyde in cacodylate buffer (0.1 m cacodylate in 1× PBS) at 4 °C overnight. Prior to embedding, cells were treated with 2% osmium tetroxide followed by dehydration in graded ethanol (60, 90, and 100%). Cells were then embedded in LX-112 epoxy plastic (Ladd Research, Williston, VT). Ultrathin sections of 80 nm were cut, mounted on uncoated copper grids, and stained with lead citrate and saturated aqueous uranyl acetate. Images were obtained with a Philips CM12 transmission electron microscope (Philips, Andover, MA) at 80 kV.

Mitochondrial Membrane Potential (ΔΨ_m) Measurement

Cells were permeabilized as described above and perfused with ICM containing TMRE (20 nm) for 15 min. Images were acquired using wide field fluorescence microscopy, and TMRE was present throughout the recording period. To monitor ΔΨ_m in isolated mitochondria and mitoplasts, 30 μl of the mitochondria or the mitoplast preparation was added to the recording chamber with 20 nm TMRE, and measurements were made using confocal microscopy as described above.

Data Collection and Analysis

In confocal microscopy experiments using a 60× objective, data from three to six intact cells or ∼100 isolated mitochondria/mitoplasts were acquired per image field. When using wide field fluorescence microscopy, data were collected using a 20× objective, enabling capture of ∼30 cells per image field. For all imaging experiments, multiple fields were acquired from each coverslip, and the data pooled from three to four independent coverslips were acquired on at least two different days. When comparison of different cell lines was required, the cells were cultured at the same density and passaged in parallel, and data were acquired on the same day. Fluorescence intensity changes were background-subtracted and normalized to the initial fluorescence value F₀ and expressed as F/F₀. Data were summarized as mean ± S.E., and differences between means were assessed using the Student's t test for unpaired comparisons. A one-way ANOVA with Fisher's least significant difference post hoc analysis was used for multiple comparisons. For all tests, the differences between means were accepted as statistically significant at the 95% level (p < 0.05).

RESULTS

Mitochondrially Localized Bcl-x_L Enhances Mitochondrial Ca²⁺ Uptake in Intact and Permeabilized MEF Cells

To investigate the role of Bcl-x_L in regulating [Ca²⁺]_mito uptake in response to ER Ca²⁺ release, wild-type (WT) and Bcl-x_L-KO cells were co-loaded with Rhod-2 and Fluo-2 to simultaneously monitor [Ca²⁺]_mito and [Ca²⁺]_cyto, respectively, and imaged using confocal microscopy (Fig. 1A). To evoke an InsP₃R-dependent Ca²⁺ release, cells were challenged with 1 mm ATP applied in zero Ca²⁺-containing buffer. Consistent with our previous observations (9), a larger amplitude InsP₃-dependent ER Ca²⁺ release was observed in Bcl-x_L-KO cells compared with WT (Fig. 1, A and B). A concomitant rise in [Ca²⁺]_mito was observed in both cell types, but surprisingly the magnitude of the [Ca²⁺]_mito uptake in Bcl-x_L-KO cells was smaller than that in WT despite the larger ER Ca²⁺ release (Fig. 1B). These data suggest that deletion of Bcl-x_L impinges on the ability of mitochondria to accumulate Ca²⁺. However, this was not due to structural or morphological changes because neither the mitochondrial biomass nor the architecture of ER-mitochondrial contact sites was different in Bcl-x_L-KO cells (Fig. 1, C–F).

FIGURE 1.

Bcl-x_L promotes ER to mitochondrial Ca²⁺ transfer without affecting ER or mitochondrial morphology. A, representative images showing intact WT and Bcl-x_L-KO cells co-loaded with Rhod-2 and Fluo-2 during stimulation with 1 mm ATP to evoke InsP₃-dependent ER Ca²⁺ release. B, representative time course plots of [Ca²⁺]_cyto and [Ca²⁺]_mito are shown. The peak amplitude (mean ± S.E.) of the [Ca²⁺]_cyto response was significantly larger (p < 0.05; Student's t test) in Bcl-x_L-KO (3.20 ± 0.36) compared with WT (1.93 ± 0.41), whereas the peak amplitude of the [Ca²⁺]_mito transient was smaller in Bcl-x_L-KO (1.83 ± 0.20) compared with WT (2.84 ± 0.38). C, WT and Bcl-x_L-KO MEFs were labeled with enhanced YFP and RFP targeted to the ER and mitochondria, respectively. Representative confocal sections show mitochondria (red), ER (green), and regions of colocalization (white). The degree of ER-mitochondrial colocalization was assessed using Manders' coefficient, and no significant difference was observed between the coefficients calculated for WT (0.49 ± 0.01) and Bcl-x_L-KO (0.48 ± 0.01) cells (p > 0.05; Student's t test). D, electron micrographs of WT and Bcl-x_L-KO cells (19,500×). Arrows indicate points of close ER-mitochondrial apposition. E, the ER-mitochondrial contacts identified that were less than or equal to 100 nm were sorted into five groups (0–20, 21–40, 41–60, 61–80, and 81–100 nm) and graphed based on the number of contacts in each group normalized to the total number of contacts less than or equal to 100 nm (wild type, 88 contacts; Bcl-x_L-KO, 92 contacts). F, the expression levels of several mitochondrial proteins were detected by Western blot. COX-IV, cytochrome c oxidase subunit IV.

To examine [Ca²⁺]_mito uptake independently of ER Ca²⁺ release, Rhod-2-loaded WT and Bcl-x_L-KO cells were digitonin-permeabilized and bathed in Ca²⁺-free ICM containing mitochondrial substrates. After equilibration, cells were exposed to Ca²⁺-containing medium, and [Ca²⁺]_mito uptake was monitored in response to a range of physiologically relevant Ca²⁺ concentrations (Fig. 2A). As expected, addition of the [Ca²⁺]_mito uptake blocker ruthenium red (10 μm) completely inhibited [Ca²⁺]_mito uptake (22). Compared with WT, the magnitude of [Ca²⁺]_mito uptake was markedly smaller in Bcl-x_L-KO cells (Fig. 2B), suggesting that enhanced [Ca²⁺]_mito uptake in Bcl-x_L-expressing cells is independent of physiological coupling between the ER and mitochondria. A similar conclusion was drawn from a second series of experiments in intact cells. Here, a step increase in [Ca²⁺]_cyto evoked by photoreleasing caged Ca²⁺ resulted in more [Ca²⁺]_mito uptake in WT compared with Bcl-x_L-KO cells (Fig. 2, C and D).

FIGURE 2.

Bcl-x_L promotes mitochondrial Ca²⁺ uptake in permeabilized cells and under different [Ca²⁺]_cyto. A, representative traces showing [Ca²⁺]_mito in permeabilized WT and Bcl-x_L-KO cells during step increases in external [Ca²⁺] from 0 to 1.25, 3.4, or 10.2 μm in the presence or absence of ruthenium red (RR; 10 μm). B, summary of Δ[Ca²⁺]_mito in response to physiological changes in external [Ca²⁺]_mito (mean ± S.E.; *, p < 0.05; ANOVA). C, intact WT and Bcl-X_L-KO cells were co-loaded with Rhod-2, Fluo-2, and o-nitrophenyl EGTA, and Ca²⁺ was uncaged by pulses of UV light ranging from 150 to 550 ms. Representative time course plots of [Ca²⁺]_cyto and [Ca²⁺]_mito are shown. D, the amplitude of the change in [Ca²⁺]_mito with respect to the amplitude of the evoked [Ca²⁺]_cyto change in WT and Bcl-x_L-KO cells. Data from individual cells were pooled and binned according to the amplitude of [Ca²⁺]_cyto transients. The corresponding mean ± S.E. [Ca²⁺]_mito response was then plotted against the mean ± S.E. [Ca²⁺]_cyto response for each bin. Differences between means of [Ca²⁺]_mito for each bin were assessed using ANOVA (*, p < 0.05). E, typical recordings of [Ca²⁺]_mito in permeabilized cells stably expressing ER (Bcl-x_L-ER) or mitochondrially targeted (Bcl-x_L-mito) in the Bcl-x_L-KO background during steps from 0 to 3 μm external [Ca²⁺]. F, summary bar graphs of the peak [Ca²⁺]_mito amplitude and the maximal rate of mitochondrial Ca²⁺ uptake (mean ± S.E.; ***, p < 0.001; ANOVA). Error bars represent S.E.

We next asked whether re-expressing Bcl-x_L could restore [Ca²⁺]_mito uptake to Bcl-x_L-KO cells. Cell lines were generated in the Bcl-x_L-KO background that stably expressed Bcl-x_L specifically targeted to either ER (Bcl-x_L-ER) or mitochondria (Bcl-x_L-mito). These cells were expanded from single clones expressing Bcl-x_L at levels similar to that detected in WT and extensively characterized in our previous study (9). Permeabilized cells loaded with Rhod-2 and equilibrated in zero Ca²⁺ medium were exposed to 3 μm Ca²⁺ to stimulate [Ca²⁺]_mito uptake (Fig. 2E). The peak amplitude of the [Ca²⁺]_mito response and the maximum Ca²⁺ uptake rate were measured (Fig. 2F). Targeting Bcl-x_L to ER in Bcl-x_L-KO cells had no effect on [Ca²⁺]_mito uptake; in contrast, uptake was promoted by re-expressing Bcl-x_L at the mitochondria (Fig. 2F). These findings demonstrate that localization of Bcl-x_L to the mitochondria, but not ER, restores [Ca²⁺]_mito uptake, recapitulating the WT.

Modulation of Mitochondrial Membrane Potential Is Not a Requirement for Bcl-x_L to Promote Mitochondrial Ca²⁺ Uptake

The electrochemical driving force for [Ca²⁺]_mito uptake depends on both the ΔΨ_m and the cytosolic-mitochondrial [Ca²⁺] gradient. Interestingly, antiapoptotic Bcl-2 proteins, including Bcl-x_L, have been shown to help maintain a hyperpolarized potential during cellular stress (23, 24). Given that [Ca²⁺]_mito uptake will drive depolarization and thus limit further Ca²⁺ accumulation (25), it is possible that Bcl-x_L promotes [Ca²⁺]_mito uptake by stabilizing ΔΨ_m and maintaining the driving force for Ca²⁺.

Resting ΔΨ_m was measured in WT and Bcl-x_L-KO cells after permeabilization and equilibration in ICM containing mitochondrial substrates and the membrane potential probe TMRE (20 nm). TMRE fluorescence was normalized to the fluorescence acquired after complete ΔΨ_m dissipation by the protonophore FCCP (10 μm). There was no significant difference in resting ΔΨ_m between WT and Bcl-x_L-KO cells (Fig. 3A). We next measured dynamic changes in ΔΨ_m by monitoring TMRE fluorescence in permeabilized WT and Bcl-x_L-KO cells. As expected, switching the external bathing medium from 0 to 3 μm Ca²⁺ evoked a depolarization that was reversed upon Ca²⁺ removal (Fig. 3B). The magnitude of depolarization evoked by 3 μm Ca²⁺, however, was larger in WT, presumably due to more Ca²⁺ accumulation in these cells. Thus, Bcl-x_L does not promote [Ca²⁺]_mito uptake by maintaining a hyperpolarized ΔΨ_m and strong Ca²⁺ driving force. Interestingly, upon Ca²⁺ removal, ΔΨ_m recovered much faster in WT compared with Bcl-x_L-KO when quantified as the halftime for recovery to base line (Fig. 3C), consistent with the hypothesis that Bcl-x_L stabilizes ΔΨ_m (see “Discussion”).

FIGURE 3.

Bcl-x_L promotes mitochondrial Ca²⁺ uptake independently of mitochondrial membrane potential. A, ΔΨ_m in WT and Bcl-x_L-KO cells assessed by TMRE fluorescence normalized to fluorescence after addition of FCCP (10 μm). Data represent mean ± S.E. (p > 0.05; Student's t test). B, representative traces of ΔΨ_m in response to the addition and removal of 3 μm [Ca²⁺]. The amplitude of the Ca²⁺-induced depolarization (ΔF/F₀ TMRE) in WT and Bcl-x_L-KO cells was 0.80 ± 0.01 and 0.75 ± 0.01, respectively (p < 0.001; Student's t test). C, the half-times for the ΔΨ_m recovery upon Ca²⁺ removal are summarized. Data represent mean ± S.E. (***p < 0.001; Student's t test). D, permeabilized cells were treated with FCCP, oligomycin, rotenone, and valinomycin to collapse ΔΨ_m. Traces are depicted showing the Ca²⁺ gradient-driven [Ca²⁺]_mito uptake and efflux during application and removal of 3 μm Ca²⁺. E, the summary bar graphs show the peak [Ca²⁺]_mito amplitude and the maximal rate of [Ca²⁺]_mito uptake in 0 ΔΨ_m (mean ± S.E.; **, p < 0.01; ***, p < 0.001; Student's t test). F, typical records showing ΔΨ_m hyperpolarization measured with TMRE in response to stepping [K⁺] from 140 to 0.1 mm in permeabilized WT and Bcl-x_L-KO cells incubated without mitochondria substrates in the presence of FCCP, oligomycin, rotenone, and valinomycin. The amplitude of the hyperpolarization (ΔF/F₀ TMRE) in WT and Bcl-x_L-KO cells was 2.61 ± 0.06 and 2.71 ± 0.04, respectively (mean ± S.E.; p > 0.05; Student's t test). G, representative traces showing [Ca²⁺]_mito during a step increase in bathing [Ca²⁺] from 0 to 3 μm when ΔΨ_m was [K⁺] gradient-driven. Under these conditions, the mean ± S.E. amplitude (ΔF/F₀) recorded in WT and Bcl-x_L-KO cells was 10.96 ± 0.19 and 8.20 ± 0.16 (p < 0.001; Student's t test), and the maximum uptake rate ((ΔF/F₀)/Δt) was 1.12 ± 0.02 in WT compared with 0.89 ± 0.02 in Bcl-x_L-KO (p < 0.001; Student's t test). Error bars represent S.E.

To further confirm that Bcl-x_L does not affect [Ca²⁺]_mito uptake by influencing ΔΨ_m, [Ca²⁺]_mito uptake was measured under conditions where the driving force was exclusively due to the Ca²⁺ gradient. Cells loaded with Rhod-2 were permeabilized and perfused in Ca²⁺-free ICM. To completely dissipate ΔΨ_m, the solution was switched to ICM without mitochondrial substrates and containing 5 μm FCCP, 8 μg ml⁻¹ oligomycin, 10 μm rotenone (26). After 2 min, the [Ca²⁺] in the bathing medium was increased to 3 μm (Fig. 3D). The amplitude and maximum rate of Ca²⁺ uptake, however, were larger in the WT group (Fig. 3E), indicating that Bcl-x_L enhances Ca²⁺ uptake when the driving force is only due to the [Ca²⁺] gradient. In addition, [Ca²⁺]_mito uptake was monitored under conditions in which ΔΨ_m was generated artificially in WT and Bcl-x_L-KO cells. Here, WT and Bcl-x_L-KO cells were permeabilized, and the ΔΨ_m was dissipated as described. The bathing [K⁺] was then decreased to 0.1 mm (KCl was substituted with choline chloride) in the presence of the K⁺ ionophore valinomycin (50 ng ml⁻¹). The ensuing outward K⁺ current rapidly hyperpolarized the ΔΨ_m as measured with TMRE, and the K⁺-driven ΔΨ_m attained a new steady state in about 50 s and remained stable for ∼30 s (Fig. 3F). Under these conditions, it would be extremely unlikely for Bcl-x_L to influence ΔΨ_m; however, the effect of Bcl-x_L on [Ca²⁺]_mito uptake persisted (Fig. 3G). These data strongly support a model in which Bcl-x_L regulation of Ca²⁺ uptake into the matrix is largely independent of any influence it may have on ΔΨ_m.

Promotion of [Ca²⁺]_mito Uptake by Bcl-x_L Is Dependent on the Integrity of the Outer Mitochondrial Membrane

The mitochondrial permeability transition pore (mPTP) is a molecular complex spanning the outer and inner membranes whose formation collapses ΔΨ_m (27). The mPTP is normally associated with pathological states, but transient mPTP formation under physiological conditions can function as a Ca²⁺ efflux pathway. Importantly, inhibiting this efflux pathway by blocking mPTP has been shown to promote [Ca²⁺]_mito uptake (28). Because Bcl-x_L also has an inhibitory effect on mPTP (29), we tested the hypothesis that Bcl-x_L mediates its effect on [Ca²⁺]_mito uptake through mPTP inhibition. Isolated mitochondria were prepared from WT and Bcl-x_L-KO cells, suspended in zero Ca²⁺ medium, loaded with Rhod-2, and imaged confocally. Consistent with the data collected using intact and permeabilized cells, the peak amplitude of the [Ca²⁺]_mito response and the maximum [Ca²⁺]_mito uptake rate were significantly larger in WT compared with Bcl-x_L-KO (Fig. 4A, control traces). Treatment with the mPTP inhibitor cyclosporine A, however, had no effect on Ca²⁺ uptake in either WT or Bcl-x_L-KO mitochondria (Fig. 4, A and B), indicating that mPTP modulation cannot account for the effect of Bcl-x_L on [Ca²⁺]_mito handling.

FIGURE 4.

Promotion of [Ca²⁺]_mito uptake by Bcl-x_L is dependent on the integrity of the outer mitochondrial membrane but not on mPTP opening. A, typical traces showing [Ca²⁺]_mito uptake monitored confocally in Rhod-2-loaded mitochondria isolated from WT and Bcl-x_L-KO cells. Extracellular [Ca²⁺] was stepped from 0 to 3 μm in the absence or presence of cyclosporine A (CsA; 1 μm). B, bar graphs summarizing (mean ± S.E.) the peak [Ca²⁺]_mito uptake and maximal uptake rate (***, p < 0.001; ANOVA). C, representative Western blot detecting uncoupling protein 3 (UCP3) and cytochrome c (cyto c) in isolated mitochondrial and mitoplast preparations. D, representative confocal sections of mitoplasts in the presence of TMRE (20 nm) before and after the addition of FCCP (10 μm). Scale bar, 2 μm. E, summary data (mean ± S.E.) depicting peak [Ca²⁺]_mito uptake and uptake rate monitored in response to a step increase in extracellular [Ca²⁺] from 0 to 3 μm in mitoplasts prepared from WT and Bcl-x_L-KO cells (p > 0.05; Student's t test). Error bars represent S.E.

It is widely accepted that Bcl-x_L resides primarily on the outer mitochondrial membrane; however, an important role for its localization at the inner membrane has recently been identified (30, 31). We therefore asked whether the effect of Bcl-x_L on matrix Ca²⁺ accumulation persisted after removal of the outer membrane. Mitoplasts were prepared, and disruption of the outer mitochondrial membrane was confirmed by the loss of the intermembrane protein cytochrome c (Fig. 4C). The inner mitochondrial membrane remained viable as indicated by the presence of the inner membrane protein uncoupling protein 3 along with an intact ΔΨ_m measured with TMRE (Fig. 4, C and D). With respect to the magnitude and maximum rate of Ca²⁺ uptake, there was no statistically significant difference between mitoplasts prepared from WT or Bcl-x_L-KO cells (Fig. 4E). These data indicate that localization to the outer mitochondrial membrane is required for Bcl-x_L to promote [Ca²⁺]_mito uptake.

The Effect of Bcl-x_L on [Ca²⁺]_mito Uptake Is Dependent on an Interaction with VDAC

The VDAC is the major permeability pathway for Ca²⁺ transfer across the outer mitochondrial membrane (27). This channel is known to directly interact with several members of the Bcl-2 family, including Bcl-x_L (19, 32–34). Based on an analysis of the binding determinants, peptides representing sections of the VDAC1 sequence were shown previously to be highly effective in disrupting the interaction between either Bcl-2 or Bcl-x_L and VDAC1 (20, 35). We adopted this approach to study the effect of VDAC1/Bcl-x_L interactions on [Ca²⁺]_mito uptake. Two peptides (N-terminal (N-ter) and L14-15) were synthesized based on previously published sequences shown to block binding between VDAC1 and Bcl-2 (35). In lysates from WT MEF cells, the interaction between VDAC1 and Bcl-x_L (detected using a GST pulldown assay) was eliminated by pretreating cells with cell-permeant N-ter and L14-15 peptides but not by pretreatment with control peptide (see Fig. 6C). In imaging studies, permeabilized cells were incubated with peptides (2 μm for 2 min) prior to stimulating [Ca²⁺]_mito uptake and remained present throughout the experiment. This effectively reduced the amplitude of [Ca²⁺]_mito uptake in WT to levels normally observed in Bcl-x_L-KO cells but had no effect on Bcl-x_L-KO cells (Fig. 5, A and B). These results strongly indicate that Bcl-x_L facilitates [Ca²⁺]_mito uptake by interacting with VDAC. To support the hypothesis that this interaction is likely to be ubiquitous, the effect of cell-permeant peptides on [Ca²⁺]_mito uptake were assessed in HeLa cells and found to have qualitatively similar effects (Fig. 5, C and D). The fact that peptides are effective in other cell types also increases confidence that observations made in the Bcl-x_L-KO MEF cells are not due to artifacts or adaptations to gene knock-out. In our previous study, we performed microarray analysis of WT and Bcl-x_L-KO cell lines and failed to find any difference in expression of calcium signaling genes, including InsP₃R isoforms (9). These microarray data were re-examined in the current study to assess VDAC expression levels. Although there was no difference between cell types in the levels of VDAC2 and -3, the ratio of WT to Bcl-x_L-KO VDAC1 mRNA was slightly decreased (0.904; p = 0.01); however, this did not translate into detectable reductions in VDAC1 protein assessed using Western blot (Fig. 5E). To further probe the effects of VDAC1-based peptides, intact cells were incubated with cell-permeant peptides (20 μm for 1 h prior to recording and present throughout), and [Ca²⁺]_mito uptake in response to InsP₃R-dependent Ca²⁺ release was recorded. [Ca²⁺]_mito uptake was decreased in WT but remained unchanged in Bcl-x_L-KO cells (Fig. 5, F and G). Peptide incubation had no effect on [Ca²⁺]_cyto in the same cells (Fig. 5, H and I).

FIGURE 6.

Bcl-x_L interacts with VDAC1 and VDAC3 to promote [Ca²⁺]_mito uptake. A, Western blot of recombinant purified Bcl-x_L pulled down by GST fusion proteins of VDAC1, -2, and -3 is shown in the upper lanes with the loading control blots of GST depicted below. B, Western blot detecting Bcl-x_L expression levels in WT MEF cells after incubation with cell-permeant control peptide or peptides based on the VDAC1 sequence (N-ter and L14-15; 20 μm for 1 h). C, Western blot detection of Bcl-x_L pulled down by GST fusion proteins of VDAC isoforms from WT MEF cell lysates pretreated with control or VDAC1-based peptides. D, summary bar graphs showing the peak [Ca²⁺]_mito uptake in permeabilized VDAC knock-out cells stepped from 0 to 3 μm [Ca²⁺] in the absence or presence of 2 μm VDAC1 peptides (mean ± S.E.; *, p < 0.05; ANOVA). Cell lines included VDAC single knockouts (VDAC1-KO, VDAC2-KO, and VDAC3-KO) and VDAC1 and -3 double knock-out (VDAC1&3-KO). E, Western blot showing Bcl-x_L shRNA knockdown in WT, VDAC1 knock-out, and VDAC1 and -3 double knock-out MEF cells. F, summary bar graphs showing the effect of Bcl-x_L knockdown on the peak [Ca²⁺]_mito uptake in permeabilized WT and VDAC knock-out cells upon stepping from 0 to 3 μm [Ca²⁺] (mean ± S.E.; *, p < 0.05; Student's t test). Error bars represent S.E.

FIGURE 5.

Peptides based on the VDAC1 sequence inhibit Bcl-x_L-dependent [Ca²⁺]_mito uptake. A and B, representative traces of [Ca²⁺]_mito in permeabilized cells stepped from 0 to 3 μm [Ca²⁺] in the absence or presence of 2 μm VDAC1 control peptide (ctrl), N-ter, or L14-15 peptide. Summary bar graphs of the peak [Ca²⁺]_mito amplitudes are shown (mean ± S.E.; ***, p < 0.001; ANOVA). C and D, representative traces of [Ca²⁺]_mito in permeabilized HeLa cells in which the bathing medium was stepped from 0 to 3 μm [Ca²⁺] in the presence of VDAC1-based peptides (2 μm) are shown in C, and summary bar graphs of the peak [Ca²⁺]_mito amplitudes are shown in D (mean ± S.E.; ***, p < 0.001; ANOVA). E, Western blot detection of VDAC1 in cell lysates of WT and Bcl-x_L-KO MEF cells. F and G, intact WT and Bcl-x_L-KO cells were incubated with 20 μm cell-permeant VDAC1 peptides, and InsP₃-dependent ER Ca²⁺ release was evoked by 1 mm ATP. Representative traces of [Ca²⁺]_mito and summary bar graphs of the peak amplitudes are shown (mean ± S.E.; *, p < 0.05; ANOVA). The respective mean ± S.E. maximum uptake rates ((ΔF/F₀)/Δt) for WT and Bcl-x_L-KO under control conditions were 0.21 ± 0.04 and 0.22 ± 0.03 (not significant; ANOVA); 0.14 ± 0.01 and 0.17 ± 0.02 in the presence of N-ter (not significant; ANOVA), and 0.13 ± 0.02 and 0.16 ± 0.02 (not significant; ANOVA) in the presence of L14-15. H and I, traces depicting the [Ca²⁺]_cyto signals measured in the same cells shown in F along with summary bar graphs of the peak [Ca²⁺]_cyto amplitudes (mean ± S.E.; *, p < 0.05; ANOVA). Error bars represent S.E.

Bcl-x_L Interacts with VDAC1 and VDAC3 to Regulate [Ca²⁺]_mito Uptake

To determine whether Bcl-x_L interacts with all three VDAC isoforms, GST fusion proteins of VDAC1, -2, and -3 were generated, and the binding of purified recombinant Bcl-x_L was assessed. GST-VDAC1 and -3 bound robustly to purified recombinant Bcl-x_L (Fig. 6A). In addition, GST-VDAC1 and -3 effectively pulled down endogenous Bcl-x_L from WT MEF cell lysates (Fig. 6C). In contrast, binding of purified recombinant Bcl-x_L to VDAC2 was barely detectable (Fig. 6A), and GST-VDAC2 was unable to pull down detectable quantities of Bcl-x_L from cell lysates (Fig. 6C). Pretreating WT MEF cells with cell-permeant N-ter and L14-15 peptides had no effect on Bcl-x_L expression levels (Fig. 6B) but completely inhibited the pulldown of Bcl-x_L by GST-VDAC1 and -3 (Fig. 6C). Collectively, these biochemical data suggest that Bcl-x_L interacts with VDAC1 and -3 and that binding is effectively disrupted by peptides based on the VDAC1 sequence.

Next we investigated the effect of Bcl-x_L and VDAC isoform interactions on [Ca²⁺]_mito uptake. WT and VDAC knock-out MEF cells, including VDAC1, VDAC2, VDAC3 single knockouts and a VDAC1 and -3 double knock-out cell line, were loaded with Rhod-2 and permeabilized, and [Ca²⁺]_mito uptake was measured as described earlier. The presence of N-ter and L14-15 peptides decreased the [Ca²⁺]_mito uptake in all cell lines except the VDAC1 and -3 double KO in which only VDAC2 was expressed. Consistent with the binding studies, these data suggest that Bcl-x_L interacts with both VDAC1 and -3 to promote [Ca²⁺]_mito uptake and that this is inhibited by disrupting the interaction using peptides based on the VDAC1 sequence. Of note, deletion of VDAC isoforms either singly or in combination did not appear to reduce [Ca²⁺]_mito uptake. This was surprising and suggests some form of adaptation (see “Discussion”).

To further verify the effect of Bcl-x_L on VDAC isoforms, particularly VDAC2, we generated stable Bcl-x_L knockdown cell lines in the MEF WT, VDAC1, and VDAC1 and -3 double KO background. Bcl-x_L knockdown was achieved by infecting cells with lentiviral particles carrying Bcl-x_L shRNA and verified by Western blot (Fig. 6E). When compared with cells generated using control shRNA, WT and VDAC1 KO cells generated with Bcl-x_L shRNA showed less [Ca²⁺]_mito uptake when permeabilized and challenged with 3 μm Ca²⁺ (Fig. 6F). As expected, knocking down Bcl-x_L had no effect on [Ca²⁺]_mito uptake in VDAC1 and -3 double KO cells, consistent with binding data showing that Bcl-x_L does not interact with the VDAC2 isoform. Taken together, these results indicate that Bcl-x_L interacts with VDAC1 and VDAC3, but not VDAC2, to regulate [Ca²⁺]_mito uptake.

DISCUSSION

It is well established that VDAC is a major permeability pathway for the transfer of Ca²⁺ across the outer mitochondrial membrane (15–17). The major finding of the current study is that this permeability pathway is tightly regulated by a protein/protein interaction between Bcl-x_L and VDAC isoforms 1 and 3. Moreover, endogenous levels of mitochondrially localized Bcl-x_L under basal conditions in the absence of apoptotic stimuli facilitates Ca²⁺ uptake into the mitochondrial matrix in response to physiological Ca²⁺ elevations.

Morphological and Structural Properties of Mitochondria Are Unaffected by the Loss of Bcl-x_L

There are several scenarios in which previously reported functions of Bcl-x_L could be invoked to explain the observed data. Bcl-x_L has been shown to regulate mitochondrial fusion, fission, and biomass (36), and mitochondrial morphology can be an important factor in shaping the Ca²⁺ signal (37). Could Bcl-x_L-dependent structural changes in mitochondria be at play in the current study? This does not seem to be the case because imaging experiments at both confocal and electron microscopic resolutions did not reveal any apparent difference between mitochondria in WT and Bcl-x_L-KO cells (Fig. 1, C–F). Moreover, in both cell types, the expression levels of key mitochondrial proteins were the same, indicating that there is no difference in the total number of mitochondria.

Regions of close contact between mitochondria and ER represent specific microdomains that enable Ca²⁺ released from ER channels to be effectively sensed by closely apposed mitochondria either by virtue of proximity or through protein/protein interactions that bridge the organelles (15, 38). Considering that Bcl-x_L interacts with both VDAC and InsP₃R, it is possible that all three proteins exist in a tertiary complex at the ER-mitochondria interface to facilitate ER-mitochondrial Ca²⁺ transfer. We failed to find any difference in the number or distance between ER-mitochondrial contact sites in WT and Bcl-x_L-KO cells, arguing against such a model; however, redistribution of proteins to the contact sites remains a possibility. Nevertheless, increased Bcl-x_L-dependent [Ca²⁺]_mito uptake persisted in permeabilized cells and isolated mitochondria but not in mitoplasts, strongly implicating a mechanism intrinsic to the outer mitochondrial membrane.

Bcl-x_L Does Not Promote Mitochondrial Ca²⁺ Uptake by Influencing mPTP or ΔΨ_m

The observations that antiapoptotic Bcl-2 members regulate the sensitivity to mPTP opening (24, 39, 40) and that physiological mPTP activation may be important in mitochondrial Ca²⁺ homeostasis (28) raise the possibility that Bcl-x_L increases [Ca²⁺]_mito uptake by inhibiting mPTP activity. In this model, [Ca²⁺]_mito uptake transiently induces the formation of the mPTP, which functions as a Ca²⁺ removal pathway; Bcl-x_L would be expected to inhibit mPTP and hence removal, thereby promoting greater matrix Ca²⁺ accumulation. However, this was not the case in the current study because pharmacological inhibition of mPTP with cyclosporin A had no effect on [Ca²⁺]_mito uptake in mitochondria isolated from either WT or Bcl-x_L-KO cells (Fig. 4).

Both Bcl-2 and Bcl-x_L have long been known to regulate ΔΨ_m by helping to maintain hyperpolarization during mitochondrial stress (39, 41, 42). Presumably, this enables a greater, more tolerable [Ca²⁺]_mito load and would be antiapoptotic because ΔΨ_m loss is a prerequisite for cytochrome c release. More recently, a series of elegant studies in neurons demonstrated that Bcl-x_L when localized to the inner mitochondrial membrane functions to inhibit an ion leak pathway that is essential to its role in ΔΨ_m stabilization (30, 31). Interestingly, Bcl-x_L knockdown in these cells was associated with increased ΔΨ_m (31). In our study, resting ΔΨ_m was similar in WT and Bcl-x_L-KO cells, although faster repolarization was observed in WT after a Ca²⁺-dependent depolarization, consistent with the role of Bcl-x_L role in ΔΨ_m stabilization. The function of Bcl-x_L as a ΔΨ_m modulator, however, cannot account for the majority of its effects on [Ca²⁺]_mito because increased [Ca²⁺]_mito uptake persisted under conditions where Bcl-x_L cannot impinge on ΔΨ_m: either when ΔΨ_m was completely collapsed (Fig. 3D) or when ΔΨ_m was generated artificially by a K⁺ gradient (Fig. 3G).

Outer Membrane-localized Bcl-x_L Facilitates Ca²⁺ Uptake into the Mitochondrial Matrix

There are two lines of evidence to support the idea that Bcl-x_L exerts the majority of its effects on [Ca²⁺]_mito uptake by acting at the outer, rather than the inner, mitochondrial membrane. First, expressing mitochondrially targeted Bcl-x_L in the Bcl-x_L-KO background recapitulated the [Ca²⁺]_mito uptake phenotype of WT cells (Fig. 2E). In these studies, Bcl-x_L was tagged with the membrane localization sequence of the listerial protein ActA, a well established approach that targets proteins to the outer membrane with cytoplasmic orientation (9, 43). Second, the increase in [Ca²⁺]_mito uptake conferred by Bcl-x_L was lost in mitoplasts prepared from mitochondria isolated from Bcl-x_L-expressing cells (Fig. 4E), effectively ruling out the possibility that Bcl-x_L functions by interacting with inner membrane proteins.

The Effect of Bcl-x_L on Mitochondrial Ca²⁺ Uptake Requires an Interaction with VDAC1 or VDAC3

Traditionally, the outer mitochondrial membrane was not thought to offer a significant permeability barrier to Ca²⁺; however, this paradigm has been challenged by studies demonstrating that it not only limits Ca²⁺ delivery to the uniporter (44) but can do so in a regulated fashion (16, 17, 26). Our study provides a novel physiological mechanism whereby the magnitude of Ca²⁺ transfer to the mitochondrial matrix is regulated by protein/protein interactions between Bcl-x_L and VDAC isoforms 1 and 3 (Figs. 5 and 6). To our knowledge, ours is also the first study to demonstrate that Bcl-x_L binds to VDAC1 and -3 but not to VDAC2. The lack of binding to VDAC2 is particularly interesting because the proapoptotic Bcl-2 protein BAK is known to interact only with VDAC2 and functions to recruit BAK to the outer mitochondrial membrane (45, 46). Our data therefore raise the possibility that the binding exclusivity and functional outcomes of the Bak/VDAC2 interaction could be facilitated by the inability of VDAC2 to interact with Bcl-x_L.

We have focused exclusively on Bcl-x_L; however, Bcl-2 is also known to bind VDAC1 (35). Despite a large degree of structural and functional homology between these two antiapoptotic proteins, it cannot be assumed that they both affect VDAC function in the same way. In discussion of the current data, therefore, it is only meaningful to consider the literature regarding the Bcl-x_L/VDAC interaction. VDAC gating involves voltage-dependent transitions between high and low conductance states that are associated with dramatic changes in selectivity. Open conformations are high conductance, anion-selective states permissive to the transfer of metabolites; closed conformations are lower conductance states favoring cation selectivity (13). Biochemical and structural studies have defined the Bcl-x_L/VDAC1 interaction (33, 34, 47); however, the functional implications are still unclear. Initial studies suggested that Bcl-x_L closed the channel as evidenced by the inhibition of radiolabeled sucrose uptake into VDAC1-containing liposomes (47). More recent electrophysiological experiments, however, have not produced a consistent set of observations with Bcl-x_L being shown to promote both the open (18) and closed (20) conformations across similar voltage ranges. Because the closed state is cation-selective and more Ca²⁺-permeable (17), our observation that Bcl-x_L facilitated [Ca²⁺]_mito uptake is consistent with a model in which Bcl-x_L promotes VDAC closing. On the other hand, Bcl-x_L-induced VDAC opening cannot be ruled out because a large conductance, cation-selective open state has been described (17, 48), and it is possible that Bcl-x_L could increase the frequency of transitions to this state. Future studies will address exactly how Bcl-x_L binding affects VDAC1 Ca²⁺ permeability.

In addition to direct effects on VDAC cation permeability, there are several other ways in which Bcl-x_L could affect [Ca²⁺]_mito uptake. The interaction between VDAC and the adenine nucleotide translocase of the inner membrane (49) provides precedent that VDAC exists in complexes spanning the outer and inner membranes. It remains to be determined whether or not VDAC directly interacts with the Ca²⁺ transport machinery on the inner membrane in a similar way, but it is an intriguing possibility. Another factor to consider is that Bcl-x_L regulates the oligomeric state of the channel. In the absence of Bcl-x_L, VDAC self-associates to form dimers, trimers, and tetramers (50), whereas binding of Bcl-x_L promotes the formation and stabilization of VDAC dimers (34). The potential physiological importance of VDAC self-association was illustrated by bilayer experiments showing that VDAC cross-linking had profound effects on channel conductance (50). Intriguingly, when compared with WT controls, we observed increased [Ca²⁺]_mito uptake in VDAC1 and VDAC3 knockouts (Fig. 6D). This is somewhat counterintuitive because it is already known that deletion of any given VDAC isoform does not result in compensatory up-regulation of the remaining isoforms in these knock-out MEF cells (51). This opens up the possibility that knocking down a specific isoform alters Ca²⁺ permeation through the remaining isoforms. Perhaps such functional compensation is driven by changes in the hetero-oligomeric makeup of VDAC multimers. However, this is pure speculation, and additional studies will be required to define intermembrane complexes and assess the effect of VDAC oligomerization and the role of individual isoforms on the Ca²⁺ permeability of the outer membrane.

The Physiological Implications for Bcl-x_L-regulated [Ca²⁺]_mito

We showed previously that Bcl-x_L functions at the ER by allosterically modulating the InsP₃R to increase the frequency of oscillating Ca²⁺ signals under resting conditions (5, 6). We now demonstrate that through its interactions with VDAC1 and VDAC3 Bcl-x_L also enables the ER-released Ca²⁺ to be more effectively taken up by mitochondria. This is physiologically significant because constitutive Ca²⁺ signaling to the mitochondrial matrix is essential for the maintenance of cellular bioenergetics (52). Moreover, the ability to maintain bioenergetic competence in the face of apoptotic stimuli is strongly antiapoptotic, and this is now recognized to be one of the mechanisms by which Bcl-x_L is protective (18, 31, 53, 54). Thus, our data suggest a role for mitochondrial Ca²⁺ signaling in bioenergetics regulated by outer membrane-localized Bcl-x_L.