Apoptosis regulation by Bcl-x(L) modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating

Abstract

Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca(2+) homeostasis. Antiapoptotic Bcl-x(L) binds to the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel to enhance Ca(2+)- and InsP(3)-dependent regulation of channel gating, resulting in reduced ER [Ca(2+)], increased oscillations of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and apoptosis resistance. However, it is controversial which InsP(3)R isoforms mediate these effects and whether reduced ER [Ca(2+)] or enhanced [Ca(2+)](i) signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP(3)R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP(3)R. In contrast, coexpression of each isoform with Bcl-x(L) conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER membranes, Bcl-x(L) increased the apparent sensitivity of all three InsP(3)R isoforms to subsaturating levels of InsP(3). Expression of Bcl-x(L) reduced ER [Ca(2+)] in type 3 but not type 1 or 2 InsP(3)R-expressing cells. In contrast, Bcl-x(L) enhanced spontaneous [Ca(2+)](i) signaling in all three InsP(3)R isoform-expressing cell lines. These results demonstrate a redundancy among InsP(3)R isoforms in their ability to sensitize cells to apoptotic insults and to interact with Bcl-x(L) to modulate their activities that result in enhanced apoptosis resistance. Furthermore, these data suggest that modulation of ER [Ca(2+)] is not a specific requirement for ER-dependent antiapoptotic effects of Bcl-x(L). Rather, apoptosis protection is conferred by enhanced spontaneous [Ca(2+)](i) signaling by Bcl-x(L) interaction with all isoforms of the InsP(3)R.

Fig. 1.

Expression of each individual InsP₃R isoform enhances apoptotic sensitivity. (A) Flow diagram depicting the strategy for generation of single clones of DT40-InsP₃R-KO stably expressing rat types 1, 2, and 3 InsP₃R or the relevant empty vector; the single colony selected is indicated by a number. (B) Expression level of each InsP₃R isoform in the selected clones was examined by Western blotting. Data are representative of three independent experiments. (C) Cell viability after treatment at time 0 with 20 μg ml⁻¹ anti-BCR antibody (anti-IgM) of two clones (red and green circles) expressing type 1, 2, or 3 InsP₃R and one clone expressing vector alone (black diamond) (same clones as in B). Data normalized to time 0 represent mean ± SEM of at least three separate trials performed in triplicate.

Fig. 2.

Each InsP₃R isoform enhances antiapoptotic efficacy of Bcl-x_L. (A) Flow diagram depicting strategy for generation of single clones expressing Bcl-x_L in a background of DT40-InsP₃R-KO-expressing empty vectors or InsP₃R type 1, 2, or 3. (B) Expression level of Bcl-x_L, InsP₃R, sarcoplasmic/ER Ca²⁺-ATPase 2 (SERCA2), and actin in the selected clones characterized by Western blotting. Data are representative of three independent experiments; the single colonies selected are indicated by a number. (C) Viability after treatment at time 0 with 20 μg ml⁻¹ anti-BCR antibody (anti-IgM) of DT40 cells expressing InsP₃R isoforms with (red open circles) or without (red filled circles) Bcl-x_L, and InsP₃R-KO cells with (black open diamonds) or without (black filled diamonds) Bcl-x_L. Data normalized to time 0 represent mean ± SEM of at least three separate trials performed in triplicate. Asterisks indicate P < 0.05 (*) or P < 0.001 (**), ANOVA at 72 and 96 h time points. Similar results were obtained by using the second set of clones [see supporting information (SI) Fig. 6].

Fig. 3.

Bcl-x_L augments single-channel activity of each InsP₃R isoform in response to low [InsP₃]. (A) Typical single-channel recordings of types 1, 2, and 3 InsP₃R in the presence of saturating (10 μM) or subsaturating (1 μM) [InsP₃] in the absence or presence of recombinant Bcl-x_L (1 μM; rBcl-x_L). Holding potential was 40 mV, upward deflections represent transition to the open state; pipette [Ca²⁺] was 2 μM. Mean ± SEM of the channel P_o under each condition are: 10 μM InsP₃ (type 1, 0.108 ± 0.04; n = 9), (type 2, 0.552 ± 0.12; n = 5), (type 3, 0.713 ± 0.09; n = 4); 1 μM InsP₃ (type 1, 0.013 ± 0.003; n = 7), (type 2, 0.021 ± 0.01; n = 6), (type 3, 0.052 ± 0.01; n = 6); 1 μM InsP₃ + 1 μM rBcl-x_L (type 1, 0.131 ± 0.06; n = 6), (type 2, 0.105 ± 0.02; n = 3), (type 3, 0.354 ± 0.08; n = 5). (B) Normalized summary of the effects of Bcl-x_L on the P_o of types 1, 2, and 3 InsP₃R. Statistical analysis was carried out before normalization; asterisks indicate P < 0.05 (*) or P < 0.01 (**), unpaired Student's t test.

Fig. 4.

Effects of Bcl-x_L interaction with InsP₃R isoforms on ER and cytoplasmic Ca²⁺ regulation. (A) Typical records depicting single-cell [Ca²⁺]_i response to application of 1 μM thapsigargin (TG) in the absence of extracellular Ca²⁺ in types 1, 2, and 3 InsP₃R-expressing DT40 cells coexpressing either Bcl-x_L (red) or vector alone (black). Each trace represents mean of 30 cells within image field. DT40 cell clones used were the same as those used in apoptosis assays. (B) Summary of the peak [Ca²⁺]_i response to TG. Data pooled from at least three independent coverslips represent mean ± SEM of no fewer than 90 cells. Asterisks indicate P < 0.001 (***), Student's unpaired t test. (C) Typical single-cell [Ca²⁺]_i transients in response to 5 μg ml⁻¹ anti-BCR antibody (anti-IgM) in InsP₃R type 1-, 2-, or 3-expressing DT40 cells coexpressing either Bcl-x_L (red) or vector alone (black). (D) Summary data represent peak amplitudes (mean ± SEM) for at least 40 cells in multiple trials. Asterisks indicate P < 0.001 (***), Student's unpaired t test.

Fig. 5.

Bcl-x_L modulates spontaneous [Ca²⁺]_i oscillations. (A) Representative traces showing spontaneous [Ca²⁺]_i oscillations in type 1, 2, or 3 InsP₃R-expressing DT40 cells coexpressing Bcl-x_L (red) or vector alone (black). (B and C) The difference in frequency (B) and no. of oscillating cells (C) (mean ± SEM) between vector-only and Bcl-x_L-expressing cells. Data are pooled from at least three independent coverslips. Asterisks indicate P < 0.001 (***), Student's unpaired t test.