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

Figures

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  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).

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  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 rIP3R1RW 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 rIP3R1RW 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 rIP3R1RW (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…

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  Figure 3. Heterotetramers of WT rIP3R1 and rIP3R1RW are predominantly nonfunctional when expressed in DT40-3KO.A, monomeric WT rIP3R1 and mutant rIP3R1RW cell lines, as well as dimeric R1/R1, R1/R1RW, R1RW/R1, and R1RW/R1RW 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 rIP3R1RW (red), R1/R1 (dark green), R1/R1RW (orange), R1RW/R1 (purple), and R1RW/R1RW (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…

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  Figure 4. rIP3R1RW is poorly functional when expressed in HEK-3KO.A, multiple WT rIP3R1, WT hIP3R1, mutant rIP3R1RW, mutant rIP3R1ND, and mutant hIP3R1RW 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 rIP3R1RW (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 rIP3R1RW (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…

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  Figure 5. rIP3R1ND 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 rIP3R1ND 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 rIP3R1ND (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 rIP3R1ND (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…

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  Figure 6. rIP3R1ND is poorly functional when expressed in HEK-3KO.A, multiple WT rIP3R1 and mutant rIP3R1ND 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 rIP3R1ND (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 rIP3R1ND 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,…

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  Figure 7. Heterotetramers of WT rIP3R1 and rIP3R1ND are predominantly nonfunctional when expressed in DT40-3KO.A, monomeric WT rIP3R1 and mutant rIP3R1ND cell lines, as well as dimeric R1/R1, R1/R1ND, and R1ND/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 rIP3R1ND (red), R1/R1 (dark green), R1/R1ND (orange), and R1ND/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…

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  Figure 8. mIP3R2GS 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 mIP3R2GS 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 mIP3R2GS 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 mIP3R2GS (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…

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  Figure 9. mIP3R2GS is predominantly nonfunctional when expressed in HEK-3KO.A, multiple WT mIP3R2 and mutant mIP3R2GS 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 mIP3R2GS (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 mIP3R2GS 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…

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  Figure 10. Heterotetramers of WT mIP3R2 and mIP3R2GS 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 mIP3R2GS cell lines, as well as dimeric R2/R2, R2/R2GS, R2GS/R2, and R2GS/R2GS 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 mIP3R2GS (red), R2/R2 (dark green), R2/R2GS (orange), R2GS/R2 (purple), and R2GS/R2GS (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…