Apoptosis regulation by Bcl-x_L modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating

Expression of Individual InsP₃R Isoforms and Their Effects on Apoptosis.

By using a variant of the chicken pre-B-cell line DT40 with all three isoforms of the InsP₃R genetically deleted (26), a series of stable lines was generated that expressed recombinant rat type 1, 2, or 3 InsP₃R (Fig. 1A). For each isoform, two clones with different levels of InsP₃R expression were selected (Fig. 1B). Ig engagement of the DT40 B-cell receptor (BCR) stimulates InsP₃-dependent [Ca²⁺]_i signaling and induces apoptosis in a process that recapitulates negative selection (26, 36). Cells were stimulated with anti-chicken IgM, and cell viability was monitored over 120 h. Expression of each individual InsP₃R isoform enhanced apoptosis sensitivity (Fig. 1C). Notably, there was a correlation between the absolute expression level of the types 1 and 2 InsP₃R (2.5- and 5-fold difference between clones, respectively) and the degree of enhanced apoptosis sensitivity (Fig. 1C). This correlation was not observed in the type 3-expressing lines, likely because of the similar levels of InsP₃R-3 protein expressed in the two clones (<1.5-fold difference between them). Of note, the enhanced apoptosis sensitivity conferred by expression of each InsP₃R isoform was most prominent during the first 48 h in a pattern similar to that reported for DT40 wild-type cells that endogenously express all three chicken InsP₃Rs (29). These data demonstrate a lack of isoform specificity in the ability of the InsP₃R to contribute to apoptosis progression and furthermore show that apoptosis sensitivity can be titrated by changes in total InsP₃R protein expression.

Effects of Bcl-x_L on Apoptosis in InsP₃R-Expressing Cell Lines.

For each InsP₃R isoform, clonal lines with the highest InsP₃R expression were transfected with Bcl-x_L or vector alone. After subcloning, InsP₃R types 1, 2, and 3 colonies stably expressing comparable Bcl-x_L protein levels were selected. Stable DT40-InsP₃R knockout (KO) cells expressing Bcl-x_L or appropriate vector controls were generated in parallel (Fig. 2 A and B). Bcl-x_L expression conferred protection during IgM-stimulated apoptosis to both InsP₃R-expressing and -nonexpressing cells. Notably, however, the degree of protection was greater in cells expressing InsP₃R. This result is similar to the InsP₃R expression-dependent effect of Bcl-x_L observed previously in DT40 cells expressing their endogenous InsP₃R (29). Importantly, this synergistic effect of Bcl-x_L and InsP₃R expression on apoptosis resistance was independent of the specific mammalian InsP₃R isoform expressed (Fig. 2C). Similar results were obtained from a second set of clones (SI Fig. 6). Therefore, the mammalian InsP₃R is required for Bcl-x_L to be fully efficacious as an antiapoptotic mediator, but the degree of protection does not depend on Bcl-x_L interactions with a particular channel isoform.

Effect of Bcl-x_L on InsP₃R Isoform Single-Channel Gating.

It was demonstrated in previous patch clamp studies that Bcl-x_L increased the sensitivity of the InsP₃R to low concentrations of InsP₃ (29). However, those studies were carried out on endogenous InsP₃R channels from the Sf9 insect cell line, which is thought to express only one isoform. This fact raises two important questions: are the functional effects of Bcl-x_L common to invertebrate and mammalian InsP₃R channels, and can Bcl-x_L differentially regulate different mammalian InsP₃R isoforms? To address these issues, the activities of single-InsP₃R channels were recorded by patch clamp electrophysiology of nuclei (37) isolated from the isoform-specific InsP₃R-expressing DT40 cell lines. The InsP₃R channel activities were first observed in the presence of saturating (10 μM) [InsP₃] and 2 μM Ca²⁺, conditions expected to evoke maximal open probability (P_o) (38). Robust gating was observed in all three channel isoforms, although the mean P_o for each isoform was different (Fig. 3A). As expected, channel activity was decreased profoundly when pipette [InsP₃] was reduced to 1 μM, consistent with the [InsP₃] dependence of channel gating (38). Despite different absolute P_o values among the individual InsP₃R channel isoforms, addition to the pipette solution of purified recombinant human Bcl-x_L [rBcl-x_L (1 μM); SI Fig. 7] dramatically increased gating activated by 1 μM InsP₃ by 5- to 10-fold in all three isoforms (Fig. 3B).

Effects of Bcl-x_L–InsP₃R Isoform Interactions on ER Ca²⁺ Homeostasis and Ca²⁺ Signaling.

These single-channel data confirm that Bcl-x_L functionally interacts with all mammalian isoforms, extending previous observations of a biochemical interaction between Bcl-x_L and all three rat InsP₃R isoforms (29). Two sets of studies were undertaken to determine the effects of these interactions on in vivo [Ca²⁺]_i signaling. First, the Ca²⁺ content of the ER was assessed by single-cell imaging of the increase in [Ca²⁺]_i in response to acute inhibition of Ca²⁺ uptake into the ER by thapsigargin (29) (Fig. 4A). As assessed by this technique, which indirectly measures the ER Ca²⁺ content, the pool size was similar in the types 1- and 3-expressing cell lines, whereas it was somewhat reduced in the cells expressing the type 2 channel isoform. Bcl-x_L expression had no effect on the thapsigargin-releasable Ca²⁺ pool in cells expressing either the type 1 or 2 isoforms whereas the Ca²⁺ response was reduced by ≈30% in the cells expressing the type 3 channel isoform (summarized in Fig. 4B).

Because BCR ligation induces apoptosis in DT40 cells through an InsP₃R-dependent pathway, we next examined [Ca²⁺]_i signals in response to a saturating concentration of anti-IgM. In all three InsP₃R isoform-expressing cell lines, anti-IgM evoked oscillatory [Ca²⁺]_i signals that were superimposed on an initial transient response (Fig. 4C). The amplitudes of the initial peak [Ca²⁺]_i responses were similar in the cells expressing the type 1 or 2 isoform. In contrast, the amplitude of the initial [Ca²⁺]_i response in the type 3-expressing cells was nearly 2-fold higher (Fig. 4D). Bcl-x_L expression was without effect on the amplitudes of the initial [Ca²⁺]_i responses in cells expressing either the type 1 or 2 InsP₃R isoform, whereas the peak amplitude of the [Ca²⁺]_i transient was reduced in the type 3-expressing cells (Fig. 4 C and D), which, interestingly, is consistent with the observed Bcl-x_L-induced reduction in the total releasable ER Ca²⁺ pool specifically in the type 3-expressing cells.

We demonstrated previously that Bcl-x_L expression enhanced the probability and frequency of spontaneous, InsP₃R-dependent [Ca²⁺]_i oscillations in resting wild-type DT40 cells (29). This effect was attributed to the observed Bcl-x_L-mediated increase in the sensitivity of InsP₃R channels to low levels of InsP₃ that might exist in unstimulated cells. Because Bcl-x_L increased the single-channel activity of all three rat channel isoforms in the presence of low [InsP₃] (Fig. 3), we hypothesized that similar spontaneous InsP₃R-dependent [Ca²⁺]_i signals would be present in the Bcl-x_L-expressing mammalian InsP₃R isoform-specific DT40 cells. Bcl-x_L expression increased the frequency of spontaneous [Ca²⁺]_i oscillations in cells expressing the type 1, 2, or 3 InsP₃R isoform (Fig. 5 A and B) and increased the number of cells exhibiting spontaneous [Ca²⁺]_i oscillations in the cells that expressed the type 2 or 3 isoform (Fig. 5C). In contrast, Bcl-x_L was without effect on either the mean amplitude of the [Ca²⁺]_i spikes or the distribution of amplitudes (data not shown). These results demonstrate that expression of any of the three mammalian InsP₃R channel isoforms individually recapitulates the observations made in wild-type DT40 cells. Furthermore, the current observations extend other reports of Bcl-2-dependent increases in [Ca²⁺]_i oscillation frequency in response to subthreshold agonist stimulation (29–31).

Cell Culture and Generation of Stable Cell Lines.

DT40 cells were maintained in suspension culture at 37°C (95/5% air/CO₂) in RPMI medium 1640 (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol) FBS/1% chicken serum/2 mM glutamine/100 units/ml penicillin/100 μg/ml streptomycin. Rat InsP₃R types 1, 2, and 3 cDNAs were cloned into the pIRES2-EGFP1 vector (Clontech, Mountain View, CA), and DT40-InsP₃R-KO cells were transfected with either InsP₃R-containing or empty vector by using a Nucleofector device according to the manufacturer's instructions (Amaxa, Gaithersburg, MD). For selection of stable clones, transfected cells were cultured for 2 weeks in the presence of 2 mg/ml G418. Transfected cells were identified based on GFP fluorescence and further subcloned by using fluorescence-activated cell sorting (BD, Franklin Lakes, NJ) into individual wells of a 96-well plate. InsP₃R expression was confirmed by Western blotting performed according to standard protocols and quantified by using infrared imaging Odyssey (LI-COR, Lincoln, NE). Selected InsP₃R-expressing clones were then transfected with Bcl-x_L in the pIRES2-DsRed2 vector or empty pIRES2-DsRed2. The selection of stable clones was essentially the same as described for InsP₃R except that in the first 2 weeks after transfection several rounds of cell sorting on DsRed fluorescence were required before subcloning.

Electrophysiology.

DT40 cells were washed twice with PBS and suspended in a nuclear isolation solution containing 150 mM KCl, 250 mM sucrose, 1.5 mM 2-mercapoethanol, 10 mM Tris·HCl, 0.05 mM PMSF, and protease inhibitor mixture (Complete; Roche Molecular Biochemicals, Indianapolis, IN) (pH 7.5). Nuclei were isolated by using a Dounce glass homogenizer and plated onto a 1-ml glass-bottomed dish containing standard bath solution: 140 mM KCl, 10 mM Hepes, and 0.5 mM BAPTA (free [Ca²⁺] ≈50 nM) (pH 7.1). The pipette solution contained 140 mM KCl, 0.5 mM ATP, 10 mM Hepes (pH 7.1). The free [Ca²⁺] in all solutions was adjusted to the desired level by the addition of an appropriate Ca²⁺ chelator, as described previously (38). His-tagged recombinant Bcl-x_L was purified and tested as described previously (ref. 29 and SI Fig. 7). Experiments were performed at room temperature. Data were acquired by using an Axopatch-1D amplifier (Molecular Devices, Sunnyvale, CA), and single-channel analysis was performed by using QuB software (State University of New York, Buffalo, NY).

Calcium Measurements and Apoptosis Assays.

DT40 cells were plated onto a glass-bottomed perfusion chamber mounted on the stage of an inverted microscope (Eclipse TE2000; Nikon, Melville, NY) and incubated with Fura-2 AM (2 μM; Molecular Probes, Eugene, OR) for 30 min at room temperature in normal culture medium. Cells were then continuously perfused with Hanks' balanced salt solution (Sigma, St. Louis, MO) containing 1.8 mM CaCl₂ and 0.8 mM MgCl₂ (pH 7.4). Fura-2 was alternately excited at 340 and 380 nm, and the emitted fluorescence filtered at 510 nm was collected and recorded by using a CCD-based imaging system running Ultraview software (PerkinElmer, Waltham, MA). Dye calibration was achieved by applying experimentally determined constants to the standard equation [Ca²⁺] = K_d·β·(R − R_min)/(R_max − R).

Cell viability was determined after application of anti-BCR antibody (20 μg ml⁻¹; Southern Biotech, Birmingham, AL) by using DAPI (1 μg ml⁻¹; Molecular Probes) or TOTO-3 staining (100 nM; Molecular Probes), and assays were performed on an LSR or Calibur flow cytometer (BD).

Analysis and Statistics.

Data were summarized as the mean ± SEM, and the statistical significance of differences between means was assessed by using unpaired t tests or one-way ANOVA with Tukey's post hoc comparison test. Differences between means were accepted as statistically significant at the 95% level (P < 0.05). Analysis of Ca²⁺ transient amplitudes was semiautomated by using macros custom-written for IGOR Pro (WaveMetrics, Lake Oswego, OR).