Disease-associated mutations in inositol 1,4,5-trisphosphate receptor subunits impair channel function

Abstract

The inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP₃Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP₃R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP₃R channel activity. Additionally, we designed concatenated IP₃R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP₃R channel function, we observed that all the mutations studied resulted in severely attenuated Ca²⁺ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP₃R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.

Keywords: Gillespie syndrome (GS); anhidrosis; calcium channel; calcium imaging; calcium intracellular release; calcium signaling; imaging; inositol 1,4,5-trisphosphate (IP3); inositol 1,4,5-trisphosphate receptor (IP3R); inositol trisphosphate receptor (InsP3R); signal transduction; spinocerebellar ataxia; spinocerebellar ataxia (SCA).

Figure 1

Linear schematic of the four domains of the IP₃R and the disease-associated IP₃R mutations found in the LBD, regulatory and coupling, and C-terminal domains.A, disease-associated mutations found in the LBD (4950, 51, 5253, 6869, 7071). B, disease-associated mutations found in the regulatory and coupling domain (47–49, 52–54, 65, 7273, 74, 75, 76, 77, 78, 79, 80, 8182). C, disease-associated mutations found in the C-terminal domain (5253, 5455, 65, 8384, 85, 86, 87, 8889). Diseases associated with mutations in at least one isoform of IP₃R include SCA29 (blue), Gillespie syndrome (orange), anhidrosis (pink), neuropathy (green), pontocerebellar hypoplasia (dark blue), spinocerebellar ataxia 15/16 (light blue), head and neck squamous cell carcinoma (red), Sézary Syndrome (purple), and familial isolated primary hyperparathyroidism (gray).

Figure 2

rIP3R1RW is nonfunctional when expressed in DT40-3KO due to decreased IP3 binding.A, Arg-269 (red) is conserved among all three human IP₃R isoforms and evolutionarily conserved in the IP₃R1 isoform. B, chimera (PDB code 3JAV) was used to visualize WT Arg-269 (yellow, left panel) and R269W mutant Trp-269 (yellow, right panel) interacting with IP₃ in the ligand binding cleft. C, WT rIP₃R1 and mutant rIP₃R1^RW cell lines were generated in the IP₃R-null DT40-3KO cells and Western blotted. D, binding of 2.5 nm [³H]IP₃ to WT rIP₃R1 and rIP₃R1^RW in the presence of increasing concentrations (0 nm, 1 nm, 3 nm, 10 nm, 30 nm, 100 nm, 300 nm, 1 μm, 3 μm, 5 μm, 10 μm, and 50 μm) of cold IP₃ in a competitive radioligand-binding assay. 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 rIP₃R1 (green), and rIP₃R1^RW (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 DT40-3KO cell line; one-way ANOVA with Tukey's test performed in F (F_19,4699= 753.0, p < 0.0001) and G (F_19,50 = 284.6, p < 0.0001).

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).

Figure 4

rIP₃R1^RW is poorly functional when expressed in HEK-3KO.A, multiple WT rIP₃R1, WT hIP₃R1, mutant rIP₃R1^RW, mutant rIP₃R1^ND, and mutant hIP₃R1^RW cell lines were generated in the IP₃R-null HEK-3KO cells and Western blotted. B, immunocytochemistry for HEK-3KO cell lines expressing either WT rIP₃R1 (left) or mutant rIP₃R1^RW (right). Top, IP₃R1 detection (green); middle, DAPI detection (blue); bottom, merged images of IP₃R1 and DAPI. Scale bars, 10 μm. C, representative traces show Ca²⁺ signals of IP₃R-null HEK-3KO cells (blue), WT rIP₃R1 (green), and rIP₃R1^RW (red) in response to trypsin (500 nm) when loaded with Fura-2/AM. D, scatter plots summarizing change in amplitude (peak ratio – basal ratio: average of initial 5 ratio points) for experiments similar to those shown in C when treated with 5, 50, and 500 nm of trypsin. Boxes represent the 25th, 50th, and 75th percentiles, whereas whiskers represent 5th and 95th percentiles and mean is represented by colored circles. E, stacked bar graph summarizing the percentage of amplitudes from D, 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 C. F, dose-response curve showing Ca²⁺ response of Fura-2/AM-loaded WT rIP₃R1, WT hIP₃R1, rIP₃R1^RW, and hIP₃R1^RW cells when treated with increasing concentrations (1 nm, 10 nm, 30 nm, 100 nm, 300 nm, 1 μm, and 3 μm) of trypsin using a Flexstation3 96-well–plate reader. Data are mean ± S.E. of three (n = 3) independent experiments. ***, p < 0.001 when compared with WT rIP₃R1 cell line and ###, p < 0.001 when compared with HEK-3KO cell line; one-way ANOVA with Tukey's test was performed in D (F_15,1352 = 407.1, p < 0.0001) and E (F_15,34 = 108.5, p < 0.0001). Unless otherwise stated, all data above comes from at least n = 3 experiments.

Figure 5

rIP₃R1^ND is nonfunctional when expressed in DT40-3KO.A, Asn-602 (red) and Thr-594 (blue) are conserved among all three human IP₃R isoforms and evolutionarily conserved in the IP₃R1 isoform. B, chimera (PDB 6MU1) was used to visualize WT Asn-602 (yellow) between two α-helical regions in the ARM1 domain (red) and adjacent to the β-TF1 and β-TF2 domains in the N terminus (blue). C, WT rIP₃R1 and several mutant rIP₃R1^ND cell lines generated in the IP₃R-null DT40-3KO cells were Western blotted. D, binding of 2.5 nm [³H]IP₃ to WT rIP₃R1 (green) and rIP₃R1^ND (red) in the presence of increasing concentrations (0 nm, 1 nm, 3 nm, 10 nm, 30 nm, 100 nm, 300 nm, 1 μm, 3 μm, 5 μm, 10 μm, and 50 μm) of cold IP₃ in a competitive radioligand binding assay. 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 rIP₃R1 (green), and rIP₃R1^ND (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 DT40-3KO cell line; one-way ANOVA with Tukey's test was performed in F (F_7,3070 = 525.9, p < 0.0001) and G (F_9,19 = 177.2, p < 0.0001).

Figure 6

rIP₃R1^ND is poorly functional when expressed in HEK-3KO.A, multiple WT rIP3R1 and mutant rIP₃R1^ND cell lines were generated in the IP₃R-null HEK-3KO cells and Western blotted. B, representative traces show Ca²⁺ signals of IP3R-null HEK-3KO cells (blue), WT rIP₃R1 (green), and rIP₃R1^ND (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 when treated with 0.5, 1, 10, and 500 nm trypsin. 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, dose-response curve showing Ca²⁺ response of Fura-2/AM-loaded WT rIP₃R1 and rIP₃R1^ND cells when treated with increasing concentrations (0.5 nm, 1 nm, 3 nm, 10 nm, 30 nm, 100 nm, 300 nm, 500 nm, 1 μm, 2.5 μm, and 5 μm) of trypsin using a Flexstation3 96-well–plate reader. Data are mean ± S.E. of three (n = 3) independent experiments. **, p < 0.01 and ***, p < 0.001 when compared with WT rIP₃R1 cell line and ###, p < 0.001 when compared with HEK-3KO cell line; one-way ANOVA with Tukey's test was performed in C (F_10,791 = 532.1, p < 0.0001) and D (F_10,22 = 108.6, p < 0.0001). Unless otherwise stated, all data above comes from at least n = 3 experiments.

Figure 7

Heterotetramers of WT rIP₃R1 and rIP₃R1^ND are predominantly nonfunctional when expressed in DT40-3KO.A, monomeric WT rIP₃R1 and mutant rIP₃R1^ND cell lines, as well as dimeric R1/R1, R1/R1^ND, and R1^ND/R1 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^ND (red), R1/R1 (dark green), R1/R1^ND (orange), and R1^ND/R1 (purple) in response to trypsin (500 nm) when loaded with Fura-2/AM. C, scatter plots summarizing the 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 the 5th and 95th percentiles and the 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, monomeric WT rIP3R1, dimeric R1/R1, trimeric R1/R1/R1, and tetrameric R1/R1/R1/ND cell lines generated in the IP3R-null DT40-3KO cells were Western blotted. F, multiple representative traces show Ca²⁺ signals of IP3R-null DT40-3KO cells expressing R1/R1/R1/R1 (green) and R1/R1/R1/ND (red) tetramers in response to trypsin (500 nm) when loaded with Fura-2/AM. G, scatter plots summarizing change in amplitude for experiments similar to those shown in F when treated with 500 nm trypsin. H, stacked bar graph summarizing the percentage of amplitudes from G, 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 F. I, traces show Ca²⁺ signals of β-escin permeabilized IP3R-null DT40-3KO cells (blue), R1/R1/R1/R1 (green), and R1/R1/R1/ND (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 rIP3R1 and rIP₃R1^ND in B–D and DT40-3KO in B–H came from Fig. 5. 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_7,3070 = 525.9, p < 0.0001), D (F_9,19 = 177.2, p < 0.0001), G (F_7,3070 = 525.9, p < 0.0001), and H (F_9,19 = 177.2, p < 0.0001).

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).

Figure 9

mIP₃R2^GS is predominantly nonfunctional when expressed in HEK-3KO.A, multiple WT mIP₃R2 and mutant mIP₃R2^GS cell lines were generated in the IP₃R-null HEK-3KO cells and Western blotted. B, representative traces show Ca²⁺ signals of IP₃R-null HEK-3KO cells (blue), WT mIP₃R2 (green), and mIP₃R2^GS (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 when treated with 500 nm trypsin. 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, dose-response curve showing Ca²⁺ response of Fura-2/AM-loaded WT mIP₃R2 and mIP₃R2^GS cells when treated with increasing concentrations (1 nm, 10 nm, 30 nm, 100 nm, 300 nm, 1 μm, and 3 μm) of trypsin using a Flexstation3 96-well–plate reader. Data are mean ± S.E. of three (n = 3) independent experiments. ***, p < 0.001 when compared with WT rIP₃R1 cell line and ###, p < 0.001 when compared with HEK-3KO cell line; one-way ANOVA with Tukey's test was performed in C (F_6,809 = 707.1, p < 0.0001) and D (F_6,17 = 335.6, p < 0.0001). Unless otherwise stated, all data above comes from at least n = 3 experiments.

Figure 10

Heterotetramers of WT mIP₃R2 and mIP₃R2^GS retain partial functionality when expressed in DT40-3KO.A, the current IP₃R structural model based on the cryo-EM structure of IP₃R1 (PDB codes 6MU1 and 6MU2) indicates that the G2498S substitution may form H bonds with neighboring Arg-2544. B, monomeric WT mIP₃R2 and mutant mIP₃R2^GS cell lines, as well as dimeric R2/R2, R2/R2^GS, R2^GS/R2, and R2^GS/R2^GS cell lines generated in the IP₃R-null DT40-3KO cells were Western blotted. C, representative traces show Ca²⁺ signals of IP₃R-null DT40-3KO cells (blue), WT mIP₃R2 (green), and mIP₃R2^GS (red), R2/R2 (dark green), R2/R2^GS (orange), R2^GS/R2 (purple), and R2^GS/R2^GS (dark red) in response to trypsin (500 nm) when loaded with Fura-2/AM. D, scatter plots summarizing change in amplitude (peak ratio – basal ratio: average of initial 5 ratio points) for experiments similar to those shown in C. Boxes represent the 25th, 50th, and 75th percentiles, whereas whiskers represent the 5th and 95th percentiles and mean is represented by colored circles. E, stacked bar graph summarizing the percentage of amplitudes from D, 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 C. F, traces show Ca²⁺ signals of β-escin permeabilized WT mIP₃R2 (green), and mIP₃R2^GS (red), R2/R2 (dark green), R2/R2^GS (orange), R2^GS/R2 (purple), and R2^GS/R2^GS (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, mIP₃R2, and mIP₃R2^GS in D–-F came from Fig. 8. *, p < 0.05 and ***, p < 0.001 when compared with corresponding WT cell line (WT mIP₃R2 for monomers and R2/R2 cell line for dimers) and ###, p < 0.001 when compared with DT40-3KO cell line; one-way ANOVA with Tukey's test was performed in D (F_9,1568 = 437.7, p < 0.0001) and E (F_9,27 = 61.27, p < 0.0001). Unless otherwise stated, all data above comes from at least n = 3 experiments. ns, non-significant.