Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: A. Comparison of CD Ca 2+ binding site in IP 3 Rs. Overlay of rIP 3 R1 and hIP 3 R3 structures, highlighting the ARM2 and HD-IP 3 R1/CD-IP 3 R3 domains that form the Ca²⁺ binding site (outlined with a dashed line). B. Zoomed-in view of the Ca-CD site (rIP 3 R1 - 8EAR/grey; hIP 3 R3 - 6DRC/tan). The highly conserved R747-rIP 3 R1/R743-hIP 3 R3, E1127-rIP 3 R1/E1122-hIP 3 R3, E1130- rIP 3 R1/E1125-hIP 3 R3 responsible for coordination of Ca 2+ are labeled. Notably, Ca²⁺ has only been observed in the 6DRC structure. C. Structural alignment of the Ca-CD site between IP 3 R subtypes and through evolution. The Ca-CD site is not conserved in RyR. D. Overlay of rIP 3 R1 (green) and rabbit RyR1 (tan) structures (7M6A). The IP 3 R CD Ca 2+ binding residues are not conserved in rabbit RyR1 structure.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Comparison, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: Generation of stable cells in HEK-293 IP 3 R null-background A. A representative western blot depicting IP 3 R1 and GAPDH protein levels in HEK-3KO, HEK WT (HEK293), hR1 endo, stable hR1 exo 69, E1128A (clones #32 and #40), and E1131A (clones #6 and #18) cell lines. B. Scatter plot showing quantification of IP 3 R1 protein levels normalized to GAPDH. Data are presented as mean±s.e.m from n=4 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ns; non-significant when compared to hR1 exo 69 cell line. C. Localization of IP 3 R1 in hR1 exo 69, E1128A #40, and E1131A #18 mutant stable cells. Scale bar, 10 µm.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Western Blot, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: E1128A mutant stable cells exhibit enhanced sensitivity to IP 3 generating agonist compared to WT cells. Representative Ca 2+ traces showing increase in cytosolic Ca 2+ levels from A. hR1 exo, and B. E1128A cells in response to stimulation with 3, 10, and 30 μM CCh. The thicker black and red traces denote the average increase in cytosolic Ca 2+ from the population. Scatter plots comparing C. changes in amplitude (peak ratio – basal ratio: average of initial 20 ratio points) and D. percentage of responding cells (amplitude changes >0.1), from hR1 exo and E1128A cells in response to stimulation with 3, 10, and 30 μM CCh. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by two-way ANOVA with Sidak test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns; non-significant. E. Pie-charts comparing cell response heterogeneity [non-responders, sustained/global Ca 2+ signals, and oscillating Ca 2+ signals (amplitude changes >0.05)] between hR1 exo 69 (n=64 cells) and E1128A #40 (n=67 cells) cell lines in response to stimulation with 3, 10, and 30 μM CCh.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: E1131A mutant stable cells exhibit enhanced sensitivity to IP 3 generating agonist compared to WT cells. Representative Ca 2+ traces showing increase in cytosolic Ca 2+ levels from A. hR1 exo and B. E1131A cells in response to stimulation with 0.3, 1, and 3 μM CCh. The thicker black and blue traces denote the average increase in cytosolic Ca 2+ from the population. Scatter plots comparing C. changes in amplitude (peak ratio – basal ratio: average of initial 20 ratio points) and D. percentage of responding cells (amplitude changes >0.1), from hR1 exo and E1131A cells in response to stimulation with 0.3, 1, and 3 μM CCh. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by two-way ANOVA with Sidak test. *p<0.05, **p<0.01, and ****p<0.0001. E. Pie-charts comparing cell response heterogeneity [non-responders, sustained/global Ca 2+ signals, and oscillating Ca 2+ signals (amplitude changes >0.05)] between hR1 exo (n=58 cells) and E1131A (n=72 cells) cell lines in response to stimulation with 0.3, 1, and 3 μM CCh.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site Ca 2+ binding mutants exhibit spontaneous Ca 2+ puffs under basal conditions. A. Maximal intensity projections of Cal-520 fluorescence at 5 s intervals from hR1 exo, E1128A, and E1131A cell lines under basal resting conditions. Elementary Ca 2+ signals in E1128A, and E1131A cells are highlighted. B. Representative traces showing fluorescence changes from the center of a single puff site (1 × 1 μm) from hR1 exo (black), E1128A (red), and E1131A (blue) cell lines. C. Scatter plots summarizing number of puff sites/cell, number of puffs/cell, and average amplitudes of the Ca 2+ puffs are shown for hR1 exo, E1128A, and E1131A cells. Data are presented as mean±s.e.m from n=10 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p<0.01. D. Representative sweeps obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs without IP 3 in the presence of either 100 µM or 5 mM ATP and 1 µM [Ca 2+ ].

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Binding Assay, Fluorescence, Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site Ca 2+ binding mutants exhibit augmented Ca 2+ puffs upon uncaging ci-IP 3 . A. Maximal intensity projections of Cal-520 fluorescence at 5 s intervals from hR1 exo, E1128A, and E1131A cell lines upon uncaging 0.05 µM ci-IP 3 . Elementary Ca 2+ signals are highlighted. E. Representative traces showing fluorescence changes upon uncaging ci-IP 3 from the center of a single puff site (1 × 1 μm) from hR1 exo (black), E1128A (red), and E1131A (blue) cell lines. Individual Ca 2+ puffs from the boxed area highlighting longer decay/fall time for the CD site Ca 2+ binding mutants when compared to hR1 exo cells. Scatter plots summarizing B. number of puff sites/cell, C. number of puffs/cell, D. average amplitudes of the Ca 2+ puffs, and F. mean-rise (r) and -fall (f) times for the fluorescence to rise/decay to various levels (0%–100%) are shown. Data are presented as mean±s.e.m from n=10 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p<0.01, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: Generation of stable cells in DT-40 3KO background. A . A representative western blot depicting IP 3 R1 and GAPDH protein levels in DT40 exo R1 (clone #1), DT40 exo R1 E1128A, DT40 exo R1 E1131A, and DT40 exo R1 CD DM (E1128/1131A) cell lines. B. Scatter plot showing quantification of IP 3 R1 protein levels normalized to GAPDH. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ns; non-significant when compared to DT40 exo R1 cell line. A comparison of intracellular global Ca 2+ signals (ΔF/F o ) between WT (DT40 R1) and CD DM cells following repeated photolysis of either C. 1 µM or D. 5 µM ci-IP 3 at indicated time points (arrows). Data are presented as mean±s.e.m from n=3 independent experiments.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site is not essential for Ca 2+ -dependent inactivation of IP 3 R1 at high [ATP]. A. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs at indicated [Ca 2+ ] stimulated with 1 µM IP 3 in the presence of 5 mM ATP. B. Representative sweeps for the CD DM and hR1 at indicated [Ca 2+ ] in the presence of 1 µM IP 3 and 5 mM ATP are shown.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site is critical for Ca 2+ -dependent inactivation of IP 3 R1 at low [IP 3 ] and [ATP]. A. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs at indicated [Ca 2+ ] stimulated with 1 µM IP 3 in the presence of 100 µM ATP. B. Representative sweeps showing higher activity from CD DM compared to R1 at inhibitory [Ca 2+ ]. C. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or CD DM mutant constructs at indicated [Ca 2+ ] stimulated with 10 µM IP 3 in the presence of 100 µM ATP. D. Representative sweeps showing higher activity from CD DM compared to R1 at inhibitory [Ca 2+ ].

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct, Activity Assay