Fig 1: Constitutive currents of MTR-GoF mutants are inhibited by CETR-LoF mutations in a dominant manner independent of their location relative to each other. Four schemes representing the location of the investigated residues within a single subunit (top left) of Orai1 or the whole channel complex (top middle), for either LoF or GoF mutation closer to the pore, respectively. The red stop sign represents the position of the LoF mutation, while the blue circle shows the position of the GoF mutation. The spheres indicate the impact of the GoF mutation on the entire subunit. Special focus was addressed to H134, L174, and S239, owing to their location in the MTR close to pore (H134) or at the channel periphery (S239) or in the CETR (L174), respectively (A–D, F–I). By combining a GoF in the MTR and an LoF mutation in the CETR at the respective positions and investigating their impact on each other, we examined whether interdependent TM domain motions within the entire channel complex are necessary for pore opening. To provide a solid foundation for the conclusions obtained with the mutations at H134, L174, and S239, we investigated a diversity of other double mutants combining distinct LoF and GoF mutations at other positions (E, J). A, time courses of current densities after whole-cell break-in of Orai1 S239C compared with Orai1 L174D S239C in the absence of STIM1. Constitutive currents of the MTR-GoF Orai1 S239C mutant are inhibited by the additional introduction of the LoF mutation L174D. B, block diagram of whole-cell current densities of Orai1 S239C, Orai1 L174D S239C in the absence (t = 0 s) and the presence (maximum current densities) of STIM1 (n = 4–12 cells; values are mean ± SD). The current densities differed statistically significantly for the different Orai1 variants (Welch-ANOVA F(3, 7.05) = 13.25, p < 0.005. Games–Howell post hoc test revealed a significant difference between the GoF mutants and the corresponding GoF–LoF double mutants (p < 0.05). This holds for paired comparisons both in the absence, both in the presence, or one in the absence and one in the presence of STIM1. C, block diagram of whole-cell current densities of Orai1 S239C compared with Orai1 L174D S239C in the presence of STIM1 OASF L251S. Activation of the LoF–GoF Orai1 L174D S239C double mutant via STIM1 OASF L251S is significantly reduced compared with that of the GoF Orai1 S239C mutant (Mann–Whitney test p < 0.05). D, intensity plots of STIM1-OASF-L251S coexpressed with Orai1 S239C compared with Orai1 L174D S239C (at 4 µm, Mann–Whitney test p < 0.05). Image series depict YFP-Orai1 S239C (as quantitative analysis of STIM1-OASF-L251S localization is shown in the corresponding intensity plot in [D], the same image series like in Figure 2D is shown for Orai1 S239C) or YFP-Orai1 L174D S239C mutants, CFP-OASF L251S and overlay (the scale bar represents 10 µm). E, block diagram of maximum whole-cell current densities of Orai1 P245L compared with Orai1 I148S P245L and Orai1 E149K P245L (Welch-ANOVA F(2, 11.94) = 14.07, p < 0.001; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]); Orai1 V181K compared with Orai1 I148S V181K and Orai1 E149K V181K (Welch-ANOVA F(2, 6.27) = 27.95, p < 0.001; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]); Orai1 A235C in comparison with Orai1 L174D A235C and Orai1 S179F A235C (Welch-ANOVA F(2, 6.44) = 6.07, p < 0.05; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]); Orai1 S239C in comparison with Orai1 S179F S239C (Welch-ANOVA F(1, 6.12) = 17.34, p < 0.01; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]); Orai1 P245L in comparison with Orai1 L174D P245L and Orai1 S179F P245L (Welch-ANOVA F(2, 9.67) = 16.06, p < 0.001; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]), all in the presence of STIM1. F, time courses of current densities after whole-cell break-in of Orai1 H134A compared with Orai1 H134A L174D in the absence of STIM1. Constitutive currents of the MTR-GoF Orai1 H134A mutant are inhibited by the additional introduction of the LoF mutation L174D. G, block diagram of whole-cell current densities of Orai1 H134A, Orai1 H134A L174D in the absence and presence of STIM1 (n = 4–12 cells; values are mean ± SD). The current densities differed statistically significantly for the different Orai1 variants (Welch-ANOVA F(3, 6.70) = 32.48, p < 0.005; Games–Howell post hoc test revealed a significant difference between the GoF mutants and the corresponding GoF-LoF double mutants [p < 0.05]). This holds for paired comparisons both in the absence, both in the presence, or one in the absence and one in the presence of STIM1). H, block diagram of whole-cell current densities of Orai1 H134A compared with Orai1 H134A L174D in the presence of STIM1 OASF L251S. Activation of the LoF–GoF Orai1 H134A L174D double mutant via STIM1 OASF L251S is significantly reduced compared with that of the GoF Orai1 H134A mutant (Mann–Whitney test p < 0.05). I, intensity plots of STIM1-OASF-L251S coexpressed with Orai1 H134A compared with Orai1 H134A L174D (at 4 µm, Mann–Whitney p < 0.05). Image series depict YFP-Orai1 H134A (as quantitative analysis of STIM1-OASF-L251S localization is shown in the corresponding intensity plot in (I), the same image series like in Figure 2I is shown for Orai1 H134A) or YFP-Orai1 H134A L174D mutants, CFP-OASF L251S, and overlay (the scale bar represents 10 µm). J, block diagram of maximum Orai1 mutant whole-cell current densities of Orai1 H134A compared with Orai1 H134A L174D, Orai1 H134A S179F (Welch-ANOVA F(2, 11.79) = 8.37, p < 0.01; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]) and Orai1 F136S compared with Orai1 F136S S179F (Welch-ANOVA F(1, 9.04) = 13.71, p < 0.01; Games–Howell post hoc test revealed significant difference for Orai1-GoF with each Orai1-GoF-LoF double point mutants [p < 0.05]), all coexpressed with STIM1. CETR, cytosolic extended transmembrane region; GoF, gain of function; LoF, loss of function; MTR, middle transmembrane region.
Fig 2: The inhibition of ER Ca2+ depletion by the Orai1 channel requires PKC activation and the presence of external [Ca2+]. (A) Time course of the [Ca2+]i response to the addition of ATP-TG for either untreated mock cells (red trace) or mock cells in the presence of 200 nM Gö 6976 (black trace) or the absence of external Ca2+ (pink trace). The same conditions were applied to cells overexpressing the O1-wt channel: no treatment (blue trace); 200 nM Gö 6976 (green trace) or in the absence of external Ca2+ (brown trace). The inset shows the time course of the initial increase in the [Ca2+]i triggered by the addition of ATP and TG. (B) Bar graphs show the average initial rate of [Ca2+]i rise for those conditions displayed in panel A. Data are the mean ± SEM. The student’s t-test for one tail was used to compare each treatment with its corresponding mock. Both conditions inhibited the increase in the rate of [Ca2+]i rise seen with O1-wt. (C) The time course of the reduction in the [Ca2+]ER induced by ATP and TG for those cells depicted in panel A. (D) Bar graphs showing the Mag-fluo-4 fluorescence at 7 min after the addition of ATP-TG. ** p < 0.01; *** p < 0.001. The number of independent experiments was n = 10 for mock cells and 5 for the rest. Bars represent the mean ± SEM.
Fig 3: ATP and TG stimulate the interaction of IP3R2 with O1-wt and O1-DD, but inhibit the interaction of IP3R2 with O1-AA. A proximity ligation assay (PLA) was carried out in cells expressing either eYFP-Orai1, Orai1-S27D/S30D (O1-DD), or Orai1-S27A/S30A (O1-AA) for detecting their interaction with IP3R2. (A) Fluorescence images show nuclei stained with DAPI, and red dots represent positive PLA for IP3R2 and Orai1 for cells expressing eYFP-O1 at resting conditions or (B) after being stimulated with ATP (10 µM) and TG (1 µM) for 3 min. (C,D) Dot PLA images for cells expressing O1-DD channels at rest and after being stimulated with ATP and TG, respectively. (E,F) PLA images for cells expressing O1-AA channels at rest and after stimulation with ATP and TG, respectively. (G) The average number of red dots per nucleus as an indicator of the IP3R2–Orai1 interaction for cells overexpressing wild type Orai1 (eYFP-O1, red bars), phosphomimetic Orai1 (O1-DD, blue bars), and phosphorylation-resistant Orai1 mutant (O1-AA, black bars). Statistical analysis was carried out with an unpaired, one-tail Student’s t-test with the corresponding control. Bars show the mean ± SEM. * p < 0.05; *** p < 0.001 from at least 50 cells from three independent experiments.
Fig 4: IR triggers Ca2+ regulated STIM1/Orai1 CRAC channel formation. (A) Distribution of endogenous Orai1 (magenta, first column) and STIM1 (green, second column) in Jurkat cells immune-stained with Alx647 and Alx488, respectively. Overlays of green and magenta images with magnification of indicated areas are shown in third and fourth columns. Fixed cells were obtained from untreated/nonirradiated control cells (top row), cells treated for 15 min with 2 µM Tg (central row), or cells 15 min after 5-Gy x-ray exposure (bottom row). (B) Probability of finding, in a population of Jurkat cells, positive clustering of STIM1/Orai1 (PSTIM1/Orai1+) after irradiation with 1.5 Gy (squares) or 5 Gy (circles). Criteria for cluster detection are specified in Materials and methods. For each condition, =282 cells were analyzed. (C) Representative confocal images of same cells with fluorescent donor molecule Orai1::eCFP (magenta, first column), acceptor molecule STIM1::eYFP (green, second column), and heatmaps of the resulting FRET signals (third column) 15 min after treatment. Images are from untreated cells (control), cells incubated with 2 µM Tg, 25 µl/ml ImmunoCult Human CD3/CD28/CD2 T-Ac, or irradiated with 5 Gy. All three treatments generate a visible FRET-signal in the PM. Scale bars, 10 µm. (D) Mean FRET signal (±SD, n = 5) from PM of cells as in C: untreated/nonirradiated control cells (crtl), cells 5 min in 2 µM Tg, 15 min in 25 µl/ml T-Ac, or 20 min after irradiation with 5 Gy. Statistical differences between treatments were analyzed by unpaired Student’s t test, and respective P values are given in the figure.
Fig 5: PLXND1 binds to ORAI1 in endocardial endothelial cells to regulate intracellular calcium levels. (A–B) Structure of predicted PLXND1 (A) and ORAI1(B). The elements of secondary structure (α-helices and β-sheets) are represented by different colors. (C) Schematic visualization of the PLXND1-ORAI1 complex, and the coupling regions were marked by red and blue solid circle respectively. (D)Three-dimensional structure showing PLXND1-ORAI1 in (C). PLXND1 and ORAI1 are represented as the grey and blue ribbon respectively. A view rotated 180° about the y axis is shown with the enlargement of interact surface in red solid circle (right). (E) Immunofluorescent staining was applied to mark PLXND1 (red) and ORAI1 (green) in EECs. White arrows represent their colocalization on the EECs. Scale bar: 20 μm. (F) Lysates from EECs were subjected to IP with IgG, anti-PLXND1, or anti-ORAI1 antibody, followed by western blotting for PLXND1 and ORAI1. PLXND1, plexinD1; EECs, endocardial endothelial cells; IP, immunoprecipitation; IB, immunoblotting; NC, negative control; WB, western blot.
Supplier Page from MilliporeSigma for Anti-ORAI1 antibody produced in rabbit