Figure 3.

Heterotetramers of WT rIP₃R1 and rIP₃R1^RW are predominantly nonfunctional when expressed in DT40-3KO.A, monomeric WT rIP₃R1 and mutant rIP₃R1^RW cell lines, as well as dimeric R1/R1, R1/R1^RW, R1^RW/R1, and R1^RW/R1^RW cell lines were generated in the IP₃R-null DT40-3KO cells and Western blotted. B, representative traces show Ca²⁺ signals of IP₃R-null DT40-3KO cells (blue), WT rIP₃R1 (green), and rIP₃R1^RW (red), R1/R1 (dark green), R1/R1^RW (orange), R1^RW/R1 (purple), and R1^RW/R1^RW (dark red) in response to trypsin (500 nm) when loaded with Fura-2/AM. C, scatter plots summarizing change in amplitude (peak ratio – basal ratio: average of initial 5 ratio points) for experiments similar to those shown in B. Boxes represent the 25th, 50th, and 75th percentiles, whereas whiskers represent 5th and 95th percentiles and mean is represented by colored circles. D, stacked bar graph summarizing the percentage of amplitudes from C, which fall into pre-determined ranges such that only those cells with an amplitude change greater than 0.1 ratio units (black portion of bars) are considered to be responding to the trypsin stimulus shown in B. E, traces show Ca²⁺ signals of β-escin permeabilized WT rIP₃R1 (green), and rIP₃R1^RW (red), R1/R1 (dark green), R1/R1^RW (orange), R1^RW/R1 (purple), and R1^RW/R1^RW (dark red) cell lines in response to IP₃ (30 μm) when loaded with Mag-Fura-2/AM. Data are mean ± S.E. of three (n = 3) independent experiments. Data for DT40-3KO, rIP₃R1, and rIP₃R1^RW in B–D were from Fig. 2. Unless otherwise stated, all data above comes from at least n = 3 experiments. ***, p < 0.001 when compared with WT rIP₃R1 cell line and ###, p < 0.001 when compared with DT40-3KO cell line; one-way ANOVA with Tukey's test was performed in C (F_19,4699 = 753.0, p < 0.0001) and D (F_19,50 = 284.6, p < 0.0001).