PMC

The Bcl-X_L–InsP₃R interaction modulates [Ca²⁺]_i signalling and mitochondrial NADH levels. (a) Spontaneous [Ca²⁺]_i oscillations in three representative DT40-WT cells stably expressing vector alone (left) or Bcl-X_L (middle), and DT40-InsP₃R-KO cells expressing Bcl-X_L (right).(b) The difference in frequency and number of oscillating cells (mean ± s.e.m.) between vector only and Bcl-X_L-expressing cells. (c) NAD(P)H fluorescence measurements from DT40-WT and DT40-InsP₃R-KO cells expressing Bcl-X_L or vector in response to 5 μg ml⁻¹ anti-BCR antibody (anti-IgM) and FCCP (2 μM). The average (± s. e.m.) resting NAD(P)H fluorescence and the change in NAD(P)H fluorescence in response to anti-IgM stimulation for four independent experiments are plotted in d. Asterisk indicates P < 0.001, ANOVA. Similar results were obtained using independent clones (see ).

Interaction of Bcl-X_L with InsP₃R. (a) Bcl-X_L binds to full-length types 1, 2 and 3 InsP₃R. Lysates from DT40-InsP₃R-KO cells stably expressing rat type 1 InsP₃R and from COS-7 cells that endogenously express type 2 and type 3 InsP₃R, were incubated with GST–Bcl-X_L, and bound InsP₃R was detected with isoform-specific antibodies (top three panels). Bottom panel: co-immunoprecipitation of endogenous Bcl-X_L and type 3 InsP₃R from COS-7 cells. (b) Domain structure of full-length InsP₃R and of its C-terminal region. (c) Bcl-X_L binds within the C terminus of InsP₃R. GST–Bcl-X_L failed to pull-down the V5-tagged 1–600 type 1 InsP₃R fragment expressed in COS-7 cells (top panel). Expression of 1–600 InsP₃R was verified by a western blot of cell lysates with V5-specific antibody (lane 3). Rat type 1 InsP₃R lacking the first 600 residues (Δ1–600 InsP₃R) expressed in COS-7 cells was successfully pulled-down along with endogenous InsP₃R-1 (middle panel). GST–Bcl-X_L effectively binds to the C-terminal 2512–2750 residues of type 1 InsP₃R (bottom panel). All western blots depicted are representative of three independent experiments.

tBid and Bax antagonize the effects of Bcl-X_L on InsP₃R channel activity. (a) tBid and Bax inhibit binding of Bcl-X_L to InsP₃R. COS-7 cell lysates were incubated with GST–Bcl-X_L in the absence or presence of recombinant tBid (2–200 nM; upper panel) or recombinant Bax (200 nM; lower panel), and bound InsP₃R was detected with a type 3 antibody. Recombinant neuronal calcium sensor-1 (NCS-1) was used to control for total protein concentration in both experiments. Data are representative of three independent experiments. (b) tBid and Bax inhibit the electrophysiological effects of Bcl-X_L. Typical current traces showing the effects of 100 nM recombinant tBid or Bax in the presence of 10 nM InsP₃ in nuclei isolated from cells transiently transfected with Bcl-X_L. Pipette [Ca²⁺] was 1 μM. (c) Addition of 100 nM tBid or Bax decreased P_o from 0.28 ± 0.06 (n = 5) to 0.07 ± 0.03 (n = 3) and 0.09 ± 0.05 (n = 3), respectively. Similarly, N_A was reduced from 0.74 ± 0.14 (n = 38) to 0.26 ± 0.10 (n = 33) in the presence of tBid and to 0.09 ± 0.05 (n = 33) in the presence of Bax. The product N_AP_o was also reduced. Asterisks indicate P < 0.001, unpaired t-test.

Effects of Bcl-X_L on InsP₃R single-channel activity. (a) Isolated nuclear preparation from Sf9 cells, which endogenously express only type 1 InsP₃R. Patch pipette approaching an isolated nucleus with an intact cell visible directly above; scale bar, 15 μM. (b) InsP₃R channel activity. Typical InsP₃R single-channel current recordings in the presence of saturating (10 μM) or low (10 nM) InsP₃; in the absence or presence of recombinant Bcl-X_L (rBcl-X_L; 1 μM); or with 10 nM InsP₃ in cells transiently transfected with Bcl-X_L. Channel activity was not evoked by rBcl-X_L (1 μM) alone. Pipette [Ca²⁺] was 1 μM, optimal for channel activity; arrow indicates zero current level. (c) Summary of the effects of Bcl-X_L on InsP₃R channel activity. In pipettes containing 10 nM InsP₃, the open probability (P_o) increased from 0.022 ± 0.001 (n = 2) to 0.61 ± 0.09 (n = 10) with addition of 1 μM rBcl-X_L (n = number of patches used in P_o determination). Similarly, when Bcl-X_L was overexpressed, P_o increased to 0.42 ± 0.05 (n = 11). Bcl-X_L also increased the number of activated channels (N_A). In 10 nM InsP₃, N_A was increased from 0.10 ± 0.07 (n = 20) under control conditions to 1.00 ± 0.16 (n = 54) and 1.18 ± 0.17 (n = 61) in the presence of recombinant or expressed Bcl-X_L, respectively. The total ER ion flux as indicated by the product N_AP_o was increased by Bcl-X_L (note log scale). Asterisks indicate P < 0.001, unpaired t-test. Both His- and Flag-tagged rBcl-X_L, generated using distinct purification protocols, were equally effective, whereas His-tagged NCS-1, which does not interact with InsP₃R (ref. ), had no effect either alone or in combination with 10 nM InsP₃ (data not shown). (d) Dependence of InsP₃R channel activity on [Ca²⁺] at the cytoplasmic face of the channel. Effect of [Ca²⁺]_i on P_o, N_A and N_AP_o in the presence of 10 μM InsP₃ (black inverted triangles), 10 nM InsP₃ (green circles), 10 nM InsP₃ + 1 μM rBcl-X_L (blue diamonds) and 10 nM InsP₃ + Bcl-X_L expression (red squares).

Interaction of Bcl-X_L with InsP₃R is essential for Bcl-X_L effects on ER Ca²⁺ regulation and inhibition of apoptosis. (a) The empty vectors pIRES2-DsRed2 or pBcl-X_L-IRES2-DsRed2 were stably expressed in DT40-WT and DT40-InsP₃R-KO cells. Expression levels of types 1 and 3 InsP₃R, Bcl-X_L and OxPhos complex IV (COXIV; subunit 1) were examined by western blot. Expression of the mitochondrial complex IV protein was unchanged in the Bcl-X_L-expressing clones. Depicted blots are representative of three independent experiments. (b) Effects of Bcl-X_L expression on the Ca²⁺ content of the ER (Ca²⁺_ER). Typical records depicting change in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) in response to application of 1 μM thapsigargin (TG) in DT40-WT and DT40-InsP₃R-KO cells stably transfected with either Bcl-X_L or vector alone. Ca²⁺_ER was indirectly estimated by single-cell imaging of the [Ca²⁺]_i responses to acute inhibition by thapsigargin of ER Ca²⁺ uptake. Each trace represents mean ± s.e.m. of at least six cells within the image field. Bar graph summarizes the effects of thapsigargin; data represent mean ± s.e.m. for at least 30 cells in multiple trials. Asterisk indicates P < 0.05, ANOVA. Mean values of resting [Ca²⁺]_i ranged from 80 to 100 nM, with no significant differences among duplicate clones of the four cell lines (data not shown). (c) [Ca²⁺]_i transients in response to 5 μg ml⁻¹ anti-BCR antibody (anti-IgM) in DT40-WT cells stably transfected with either Bcl-X_L or vector alone. Summary data represent the peak amplitude (mean ± s.e.m.) for at least 30 cells in multiple trials. (d) Cell viability after treatment with 20 μg ml⁻¹ anti-BCR antibody (anti-IgM) (time 0) of DT40-WT (solid symbols) and DT40-InsP₃R-KO (open symbols) cells stably expressing Bcl-X_L (red) or vector alone (same clones as in b). Similar results were obtained using independent clones (see ).