Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca2+ channels and activation of store-operated Ca2+ entry

Abstract

Mutations in all subtypes of the inositol 1,4,5-trisphosphate receptor Ca²⁺ release channel are associated with human diseases. In this report, we investigated the functionality of three neuropathy-associated missense mutations in IP₃R3 (V615M, T1424M, and R2524C). The mutants only exhibited function when highly over-expressed compared to endogenous hIP₃R3. All variants resulted in elevated basal cytosolic Ca²⁺ levels, decreased endoplasmic reticulum Ca²⁺ store content, and constitutive store-operated Ca²⁺ entry in the absence of any stimuli, consistent with a leaky IP₃R channel pore. These variants differed in channel function; when stably over-expressed the R2524C mutant was essentially dead, V615M was poorly functional, and T1424M exhibited activity greater than that of the corresponding wild-type following threshold stimulation. These results demonstrate that a common feature of these mutations is decreased IP₃R3 function. In addition, these mutations exhibit a novel phenotype manifested as a constitutively open channel, which inappropriately gates SOCE in the absence of stimulation.

Keywords: Cell biology; Molecular biology; Molecular neuroscience.

Graphical abstract

Figure 1

Linear-schematic of disease-associated mutations in the four main functional domains of the IP₃R3 Disease-associated mutations in IP₃R3 have been identified in the suppressor domain, regulatory and coupling domain, and C-terminal channel domain. These diseases include head and neck squamous cell carcinoma (red), neuropathy (green), immunopathy (blue), and immunopathy and neuropathy (purple). One family with mutations in IP₃R3 exhibited multiple diseases including chronic fatigue syndrome, small fiber neuropathy, and IgG3 deficiency (pink).

Figure 2

hR3 V615M is a poorly functional Ca²⁺ channel with elevated basal cytosolic [Ca²⁺] (A) Chimera (PDB: 6DR0) was used to visualize Val615 (yellow) in ARM1 of the regulatory and coupling domain (red) of IP₃R3. (B) Cell lines with varying expression of human IP₃R3 harboring the V615M mutation (hR3 V615M) were generated in IP₃R-null HEK-3KO cells and western blotted alongside WT cell lines, either endogenously expressed hIP₃R3 (Endo. hR3) and exogenously expressed hIP₃R3 (Exo. hR3). (C) Quantification of expression of hIP₃R3 expression with respect to GAPDH in HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 V615M (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 Ca²⁺ signals in fura-2 loaded cells in response to the addition of indicated concentrations of CCh. (E) Scatterplot summarizing the basal Ca²⁺ (average of the initial 20,340/380 ratio points in Ca²⁺-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 CCh in single-cell imaging experiments similar to those in (D). (G) Dose-response curve showing the change in amplitude of Ca²⁺ signals of Fura-2/AM loaded cell lines when treated with increasing [CCh] using a FlexStation3 96-well plate reader. (H) Representative traces of Ca²⁺ puffs recorded by TIRFM in Exo. hR3 or hR3 V615M. Uncaging of ci-IP₃ at the arrowhead. (I) Number of Ca²⁺ puff sites per cell in Exo. hR3 (green) and hR3 V615M (red) cell lines loaded with Cal-520 in TIRFM experiments and treated with1 μM ci-IP₃. (J) Number of Ca²⁺ puff per cell in Exo. hR3 (green) and hR3 V615M (red) cell lines loaded with Cal-520 in TIRFM microscopy experiments and treated with 1 μM ci-IP₃. All data are mean ± SEM of at least three (N = 3) independent experiments. ^###p < 0.001 when compared to HEK-3KO; ^tttP < 0.001, ^ttP < 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 V615M cell lines; one-way ANOVA with Tukey’s test performed in E, F and two-tailed t-test performed in (I andJ).

Figure 3

hR3 V615M cell lines exhibited depleted ER [Ca²⁺] and SOCE in the absence of agonist stimulation (A) Representative traces of changes in cytosolic [Ca²⁺] following removal of extracellular Ca²⁺ in HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 V615M (pink, orange, red) cell lines loaded with Fura-2/AM. Cells were subsequently treated with 30 μM CPA allowing the measurement of the ER Ca²⁺ store content. (B) Scatterplot summarizing the change in the basal 340/380 ratio following removal of extracellular Ca²⁺ 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 Ca²⁺ ratio (Figure 2E) 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 Ca²⁺) in experiments similar to those in (A). (D) Representative traces of Ca²⁺ signals in fura-2 loaded cells 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 Ca²⁺ ratio (Figure 2E) 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. ^###p < 0.001 when compared to HEK-3KO; ^tttP < 0.001, ^ttP < 0.01 when compared to Endo. hR3; ∗∗∗p < 0.001, ∗∗p < 0.01 when compared to Exo. hR3; and ∗∗∗(red)p < 0.001 when compared to other stably expressed hR3 V615M cell lines; one-way ANOVA with Tukey’s test performed in (B and E).

Figure 4

hR3 T1424M increased Ca²⁺ channel function with elevated basal cytosolic [Ca²⁺] (A) Chimera (PDB: 6DR0) was used to visualize Thr1424 (yellow) in the regulatory and coupling domain (red) of IP₃R3. (B) Cell lines containing varying expression of human IP₃R3 harboring the T1424M mutation (hR3 T1424M) were generated in IP₃R-null HEK-3KO cells and western blotted alongside WT cell lines; Endo. hR3 and Exo. hR3. (C) Quantification of hIP₃R expression with respect to GAPDH of HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 T1424M (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 Ca²⁺ signals in cells expressing the indicated variant in response to the addition of increasing [CCh]. (E) Scatterplot summarizing the basal Ca²⁺ (average of the initial 20,340/380 ratio points in Ca²⁺-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). (G) Dose-response curve showing the change in amplitude of Ca²⁺ signals when treated with increasing [CCh] using a FlexStation3 96-well plate reader. (H) Representative traces of Ca²⁺ puffs in the absence of stimulation in Exo. hR3 and hR3 T1424M expressing cells. (I) Number of Ca²⁺ puff sites and Ca²⁺ puffs per cell in Exo. hR3 (green) and hR3 T1424M #204 (red) cell lines loaded with Cal-520 in TIRFM microscopy experiments without photo release of ci-IP₃. (J) Number of Ca²⁺ puff sites and Ca²⁺ puffs per cell in Exo. hR3 (green) and hR3 T1424M (red) cell lines loaded with Cal-520 in TIRFM microscopy experiments following photo-release of 0.1 μM ci-IP₃. 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, F repeated from Figure 2 above. ^###p < 0.001, ^##p < 0.01 when compared to HEK-3KO; ^tttP < 0.001, ^ttP < 0.01, ^tP < 0.05 when compared to Endo. hR3; ∗∗∗p < 0.001 when compared to Exo. hR3; and ∗∗∗(red)p < 0.001, ∗∗(red)p < 0.01 when compared to other stably expressed hR3 T1424M cell lines; one-way ANOVA with Tukey’s test performed in (E and F) and two-tailed t-test performed in (I and J).

Figure 5

hR3 T1424M cell lines exhibited depleted ER [Ca²⁺] and SOCE in the absence of agonist stimulation (A) Representative traces of changes in cytosolic [Ca²⁺] following removal of extracellular Ca²⁺ in HEK-3KO (blue), Endo. hR3 (purple), Exo. hR3 (green), and hR3 T1424M (pink, orange, red) cell line. Cells were subsequently treated with 30 μM CPA allowing the measurement of the ER Ca²⁺ store content. (B) Scatterplot summarizing the change in the basal 340/380 ratio following removal of extracellular Ca²⁺ 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 Ca²⁺ ratio (Figure 2E) 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 Ca²⁺) in experiments similar to those in (A). (D) Representative traces of Ca²⁺ signals in response to the addition of 10 μM GSK-7975a when loaded with Fura-2/AM. (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 Ca²⁺ ratio (Figure 2E) 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 Figure 3 above. ^###p < 0.001 when compared to HEK-3KO; ^tttP < 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 T1424M cell lines; one-way ANOVA with Tukey’s test performed in (B and E).

Figure 6

hR3 R2524C exhibited absent Ca²⁺ channel function and an elevated basal cytosolic [Ca²⁺] (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 IP₃R3 monomers (blue). (B) Cell lines with varying expression of human IP₃R3 harboring the R2524C mutation (hR3 R2524C) were generated in IP₃R-null HEK-3KO cells and western blotted alongside WT cell lines – Endo. hR3 and Exo. hR3. (C) Quantification of expression of hIP₃R3 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 Ca²⁺ signals from the indicated cell lines in response to the addition of increasing [CCh]. (E) Scatterplot summarizing the basal Ca²⁺ (average of the initial 20,340/380 ratio points in Ca²⁺-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 4 above. ^###p < 0.001 when compared to HEK-3KO; ^tttP < 0.001, ^ttP < 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).

Figure 7

hR3 R2524C cell lines exhibited depleted ER [Ca²⁺] and SOCE in the absence of agonist stimulation (A) Representative traces of changes in cytosolic [Ca²⁺] following removal of extracellular Ca²⁺ 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 Ca²⁺ store content. (B) Scatterplot summarizing the change in the basal 340/380 ratio following removal of extracellular Ca²⁺ 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 Ca²⁺ ratio (Figure 2E) 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 Ca²⁺) in experiments similar to those in (A). (D) Representative traces of Ca²⁺ 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 Ca²⁺ ratio (Figure 2E) 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 5 above. ^###p < 0.001 when compared to HEK-3KO; ^tttP < 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).