Figure 8.

mIP₃R2^GS is nonfunctional when expressed in DT40-3KO.A, Gly-2498 (red) is conserved among all three human IP3R isoforms and evolutionarily conserved in the IP₃R2 isoform. B, chimera (PDB 6MU1) was used to visualize WT Gly-2498 (yellow) in the selectivity filter (blue) of two monomers, just prior to the 6th transmembrane (TM) domain (red). C, WT mIP₃R2 and mutant mIP₃R2^GS cell lines were generated in the IP₃R-null DT40-3KO cells and Western blotted. D, binding of 2.5 nm [³H]IP₃ to WT mIP₃R2 and mIP₃R2^GS in the presence of a maximal concentration of 50 μm cold IP₃ in a competitive radioligand binding assay (p = 0.9077). Data are mean ± S.E. of three (n = 3) independent experiments. E, representative traces show Ca²⁺ signals of IP3R-null DT40-3KO cells (blue), WT mIP₃R2 (green), and mIP₃R2^GS (red) in response to trypsin (500 nm) when loaded with Fura-2/AM. F, scatter plots summarizing change in amplitude (peak ratio – basal ratio: average of initial 5 ratio points) for experiments similar to those shown in E. Boxes represent the 25th, 50th, and 75th percentiles, whereas whiskers represent 5th and 95th percentiles and mean is represented by colored circles. G, stacked bar graph summarizing the percentage of amplitudes from F, 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 E. 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 HEK-3KO cell line; unpaired t test was performed in D and one-way ANOVA with Tukey's test was performed in F (F_9,1568 = 437.7, p < 0.0001) and G (F_9,27 = 61.27, p < 0.0001).