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.