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. 2013 Jul 5;288(27):19870-81.
doi: 10.1074/jbc.M112.448290. Epub 2013 May 17.

An interaction between Bcl-xL and the voltage-dependent anion channel (VDAC) promotes mitochondrial Ca2+ uptake (V体育官网入口)

Huiya Huang  1 , Xiangxin Hu, Colins O Eno, Guoping Zhao, Chi Li, Carl White
Affiliations

An interaction between Bcl-xL and the voltage-dependent anion channel (VDAC) promotes mitochondrial Ca2+ uptake (VSports)

Huiya Huang et al. J Biol Chem. .

"V体育安卓版" Abstract

The role of the antiapoptotic protein Bcl-xL in regulating mitochondrial Ca(2+) ([Ca(2+)]mito) handling was examined in wild-type (WT) and Bcl-xL knock-out (Bcl-xL-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca(2+) transients that produced a larger [Ca(2+)]mito uptake in WT cells compared with Bcl-xL-KO. In permeabilized cells, stepping external [Ca(2+)] from 0 to 3 μm also produced a larger [Ca(2+)]mito uptake in WT; moreover, the [Ca(2+)]mito uptake capacity of Bcl-xL-KO cells was restored by re-expression of mitochondrially targeted Bcl-xL. Bcl-xL enhancement of [Ca(2+)]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 Ca(2+) permeability pathway that directly interacts with Bcl-xL. Bcl-xL interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-xL binding. Peptides reduced [Ca(2+)]mito uptake in WT but were without effect in Bcl-xL-KO cells. In addition, peptides reduced [Ca(2+)]mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-xL interacts functionally with VDAC1 and -3 but not VDAC2 VSports手机版. Thus, an interaction between Bcl-xL and VDAC promotes matrix Ca(2+) accumulation by increasing Ca(2+) transfer across the outer mitochondrial membrane. .

Keywords: Bcl-2 Proteins; Calcium Signaling; Imaging; Mitochondria; Protein-Protein Interactions; Voltage-dependent Anion Channel V体育安卓版. .

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Figures

FIGURE 1.
FIGURE 1.
Bcl-xL promotes ER to mitochondrial Ca2+ transfer without affecting ER or mitochondrial morphology. A, representative images showing intact WT and Bcl-xL-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-xL-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-xL-KO (1.83 ± 0.20) compared with WT (2.84 ± 0.38). C, WT and Bcl-xL-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-xL-KO (0.48 ± 0.01) cells (p > 0.05; Student's t test). D, electron micrographs of WT and Bcl-xL-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-xL-KO, 92 contacts). F, the expression levels of several mitochondrial proteins were detected by Western blot. COX-IV, cytochrome c oxidase subunit IV.
FIGURE 2.
FIGURE 2.
Bcl-xL promotes mitochondrial Ca2+ uptake in permeabilized cells and under different [Ca2+]cyto. A, representative traces showing [Ca2+]mito in permeabilized WT and Bcl-xL-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-XL-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-xL-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-xL-ER) or mitochondrially targeted (Bcl-xL-mito) in the Bcl-xL-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.
FIGURE 3.
FIGURE 3.
Bcl-xL promotes mitochondrial Ca2+ uptake independently of mitochondrial membrane potential. A, ΔΨm in WT and Bcl-xL-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 ΔΨm 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-xL-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 ΔΨm 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 ΔΨm. 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 ΔΨm (mean ± S.E.; **, p < 0.01; ***, p < 0.001; Student's t test). F, typical records showing ΔΨm hyperpolarization measured with TMRE in response to stepping [K+] from 140 to 0.1 mm in permeabilized WT and Bcl-xL-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-xL-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 ΔΨm was [K+] gradient-driven. Under these conditions, the mean ± S.E. amplitude (ΔF/F0) recorded in WT and Bcl-xL-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-xL-KO (p < 0.001; Student's t test). Error bars represent S.E.
FIGURE 4.
FIGURE 4.
Promotion of [Ca2+]mito uptake by Bcl-xL 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-xL-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-xL-KO cells (p > 0.05; Student's t test). Error bars represent S.E.
FIGURE 5.
FIGURE 5.
Peptides based on the VDAC1 sequence inhibit Bcl-xL-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-xL-KO MEF cells. F and G, intact WT and Bcl-xL-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-xL-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.
FIGURE 6.
FIGURE 6.
Bcl-xL interacts with VDAC1 and VDAC3 to promote [Ca2+]mito uptake. A, Western blot of recombinant purified Bcl-xL 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-xL 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-xL 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-xL 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-xL 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.
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