Journal: iScience

Article Title: Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca 2+ channels and activation of store-operated Ca 2+ entry

doi: 10.1016/j.isci.2022.105523

Figure Lengend Snippet: hR3 R2524C exhibited absent Ca 2+ channel function and an elevated basal cytosolic [Ca 2+ ] (A) Chimera (PDB: 6DR0 ) was used to visualize Arg2524 (yellow) at the junction of the 6 th TM (purple) and LNK domain (green) in the channel pore near the negatively charged Asp2518 of neighboring IP 3 R3 monomers (blue). (B) Cell lines with varying expression of human IP 3 R3 harboring the R2524C mutation (hR3 R2524C) were generated in IP 3 R-null HEK-3KO cells and western blotted alongside WT cell lines – Endo. hR3 and Exo. hR3. (C) Quantification of expression of hIP 3 R3 with respect to GAPDH, in HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 R2524C (pink, orange, red) cell lines. Colored lines represent the mean of at least n = 3 experiments, and error bars represent SEM. Averages were normalized to that of the Endo. hR3 cell line. (D) Representative traces of Ca 2+ signals from the indicated cell lines in response to the addition of increasing [CCh]. (E) Scatterplot summarizing the basal Ca 2+ (average of the initial 20,340/380 ratio points in Ca 2+ -containing media) from experiments similar to those in (D). (F) Scatterplot summarizing change in amplitude (Peak 340/380 ratio – Basal 340/380 ratio (E)) of cell lines in response to increasing [CCh] in single-cell imaging experiments similar to those in (D). All data are mean ± SEM of at least three (N = 3) independent experiments. Control HEK-3KO (blue), Endo. hR3 (purple), and Exo. hR3 (green) data in (E and F) repeated from Figures 2 and above. ### p < 0.001 when compared to HEK-3KO; ttt P < 0.001, tt P < 0.01 when compared to Endo. hR3; ∗∗∗p < 0.001 when compared to Exo. hR3; and ∗∗∗(red)p < 0.001 when compared to other stably expressed hR3 R2524C cell lines; one-way ANOVA with Tukey’s test performed in (E and F).

Article Snippet: hIP 3 R3 R2524C , Julika Neumann (KU Leuven) , N/A.

Techniques: Expressing, Mutagenesis, Generated, Western Blot, Imaging, Control, Stable Transfection

Journal: iScience

Article Title: Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca 2+ channels and activation of store-operated Ca 2+ entry

doi: 10.1016/j.isci.2022.105523

Figure Lengend Snippet: hR3 R2524C cell lines exhibited depleted ER [Ca 2+ ] and SOCE in the absence of agonist stimulation (A) Representative traces of changes in cytosolic [Ca 2+ ] following removal of extracellular Ca 2+ in HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 R2524C (pink, orange, red) cell lines. Cells were subsequently treated with 30 μM CPA allowing the measurement of the ER Ca 2+ store content. (B) Scatterplot summarizing the change in the basal 340/380 ratio following removal of extracellular Ca 2+ in experiments similar to those in (A). Colored lines represent the mean of at least n = 3 experiments, and error bars represent SEM. (C) Scatterplot summarizing the correlation between an elevated basal 340/380 Ca 2+ ratio ( Figure 2 E) and the change in 340/380 ratio following treatment with 30 μM CPA (maximum CPA-induced amplitude – basal 340/380 ratio following removal of extracellular Ca 2+ ) in experiments similar to those in (A). (D) Representative traces of Ca 2+ signals in the indicated cell lines in response to the addition of 10 μM GSK-7975a. (E) Scatterplot summarizing the change in 340/380 ratio following the addition of GSK-7975a (average of 20,340/380 ratio points prior to GSK-7975a addition – average of 20,340/380 ratio points following 200 s of GSK-7975a addition) from experiments similar to those in (D). (F) Scatterplot summarizing the correlation between an elevated basal 340/380 Ca 2+ ratio ( Figure 2 E) and the change in 340/380 ratio following treatment with 10 μM GSK-7975a (E) in experiments similar to those in (D). All data are mean ± SEM of at least three (N = 3) independent experiments. Control HEK-3KO (blue), Endo. hR3 (purple), and Exo. hR3 (green) data in (B, C, and E), and F repeated from Figures 3 and above. ### p < 0.001 when compared to HEK-3KO; ttt P < 0.001 when compared to Endo. hR3; ∗∗∗p < 0.001 when compared to Exo. hR3; and ∗∗∗(red)p < 0.001 when compared to other stably expressed hR3 R2524CM cell lines; one-way ANOVA with Tukey’s test performed in (B and E).

Article Snippet: hIP 3 R3 R2524C , Julika Neumann (KU Leuven) , N/A.

Techniques: Control, Stable Transfection

Journal: iScience

Article Title: Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca 2+ channels and activation of store-operated Ca 2+ entry

doi: 10.1016/j.isci.2022.105523

Figure Lengend Snippet:

Article Snippet: hIP 3 R3 R2524C , Julika Neumann (KU Leuven) , N/A.

Techniques: Purification, Transduction, Recombinant, Modification, Protease Inhibitor, Software, Imaging, Microscopy, Fluorescence, Inverted Epifluorescence