Figure 6.

Mutation of key Orai1 channel pore residues in concatenated dimers. A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^ko cells expressing similar levels of the C-terminal tdT-tagged Orai1 dimers OO (Orai1-WT/Orai1-WT), OC (Orai1-WT/Orai1-V102C), (Orai1-V102C/ Orai1-WT), or CC (Orai1-V102C/Orai1-V102C). Constitutive Ca²⁺ entry was measured after addition of 1 mm Ca²⁺ (arrows) after Ca²⁺-free medium. Traces (means ± S.E.) are representative of three independent experiments. B, whole-cell patch–clamp recording of HEK-O1^ko cells expressing CC, OC, or CO; current at −100 mV was plotted against time. Cytosolic Ca²⁺ was maintained at 90 nm with BAPTA to prevent store depletion. Constitutive current was measured in 20 mm Ca²⁺ external solution, followed by blockade with 10 μm Gd³⁺. C, representative I/V curves for the constitutive currents of the HEK-O1^ko cells expressing OC, CO, and CC shown in B. D, representative I/V curves for Ca²⁺ currents following store depletion with 20 mm BAPTA in HEK-O1^koS1⁺ cells transiently expressing OC, CO, and CC. E, Ca²⁺ add-back measurements in fura-2–loaded HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdT-tagged Orai1 dimers OO (Orai1-WT/Orai1-WT), OW (Orai1-WT/Orai1-R91W), or WO (Orai1-R91W/Orai1-WT). Ca²⁺ stores were released with 2.5 μm ionomycin (iono) in Ca²⁺-free medium followed by 1 mm Ca²⁺ (arrows). Traces (means ± S.E.) are the results for all cells in three independent experiments. F, summary scatter plots of peak Ca²⁺ entry normalized to Ca²⁺ entry with WT dimer (OO); results are means ± S.E. for all cells in three independent experiments represented by E. G and H, Ca²⁺ add-back measurements using fura-2-loaded HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdT-tagged Orai1-K85E homodimers (EE) or the K85E heterodimers (OE and EO). Ca²⁺ stores were released with 2.5 μm ionomycin in Ca²⁺-free medium followed by 1 mm Ca²⁺ (arrows). Traces (means ± S.E.) are representative of all cells in three independent experiments. I, summary scatter plots of peak Ca²⁺ entry normalized to WT (OO) dimer Ca²⁺ entry; results are means ± S.E. of three independent experiments represented in G and H. J and K, representative I/V relationship of Ca²⁺ currents after store depletion in HEK-O1^koS1⁺ cells transiently expressing OE (J) or EO (K). L, scatter plots of E-FRET between stably expressed STIM1-YFP and transiently expressed C-terminally CFP-tagged Orai1 dimers (OO, OE, EO, or EE, respectively) in HEK-O1^koS1⁺ cells. E-FRET was measured at 5 min after addition of 2.5 μm ionomycin to deplete stores. **, p < 0.001. Results are means ± S.E. of three independent experiments.