Figure 5

BH4-Bcl-2 and BH4-Bcl-Xl differ in one critical amino acid, which determines their effect on the IP₃R. (a) Aligned sequence of the BH4 domain of Bcl-2 and Bcl-Xl with indication of the surface-accessible residues (in color) is shown. The critical residue Lys17 in BH4-Bcl-2 is not conserved in BH4-Bcl-Xl (depicted in blue). The sequences of these BH4-domain peptides are indicated. (b) A typical experiment of a unidirectional ⁴⁵Ca²⁺-flux assay in permeabilized MEF cells, comparing the effect of BH4-Bcl-2, BH4-Bcl-Xl, BH4-Bcl-2 K/D and BH4-Bcl-Xl D/K (80 μM of peptides). (c) The quantification of the effects of the different BH4-domain peptides (80 μM) on IICR was obtained from three to four independent experiments. Data points represent mean±S.E.M. ^*BH4-Bcl-2 and BH4-Bcl-Xl D/K are statistically different from control. ^$ BH4-Bcl-2 K/D is statistically different from BH4-Bcl-2. ^#BH4-Bcl-Xl D/K is statistically different from BH4-Bcl-Xl. (d) A dose-response curve for the different BH4-domain peptides was obtained from three independent experiments. Values are plotted as mean±S.E.M. (e) Representative IP₃R-mediated Ca²⁺ traces in intact C6 glioma cells using caged IP₃ and UV photoliberation. (f) Quantitative analysis of the area under the curve, of the traces in (e), was obtained from at least six independent experiments and data are plotted as mean±S.E.M. BH4-Bcl-2 and BH4-Bcl-Xl D/K significantly inhibited IICR, in contrast to BH4-Bcl-2 K/D and BH4-Bcl-Xl. ^*BH4-Bcl-2 and BH4-Bcl-Xl D/K are statistically different from control. ^$BH4-Bcl-2 K/D is statistically different from BH4-Bcl-2. ^#BH4-Bcl-Xl D/K is statistically different from BH4-Bcl-Xl. (g) Similar experiment as in (e and f), except that Ca²⁺ signals were elicited by ATP (1 μM). Area under the curve of different control cells was determined and set at 100%. (h) Similar experiment as in (e and f), except that cells were pretreated for 1 min with EGTA (1 mM) and exposed to thapsigargin (TG, 2.5 μM). Area under the curve for control was determined and set at 100%. Statistically significant differences were considered at P<0.05 (single symbols), P<0.01 (double symbols) and P<0.001 (triple symbols). (i) Quantitative analysis of data obtained from three independent SPR experiments, in which the binding of different concentrations of GST-Dom3 to immobilized biotin-BH4-Bcl-2 was compared with its binding to immobilized biotin-BH4-Bcl-2 K/D. The maximal signal (resonance units) during the association phase was used in this analysis. (j) Quantitative analysis of data obtained from SPR experiments, in which the binding of GST-Dom3 to biotin-BH4-Bcl-Xl was compared with its binding to biotin-BH4-Bcl-Xl D/K. The maximal signal (resonance units) during the association phase was used in this analysis