Figure 4.

The Mechanical Interplay between K157 and E245 in dOrai Has Implications for Orai-STIM Interaction

(A) The salt bridge remains intact during gating.

(B–D) In HEK STIM1-YFP cells, the effects of transiently expressed hOrai1-K85E or hOrai1-K85E-E173K mutation were examined. (B) Left, typical SOCE responses; right, mean I-V relationships measured at the peak of whole-cell current (n ≥ 5 for each condition). Mean SOCE: 16.18 ± 0.58 (n = 58) for wild-type [WT], 1.30 ± 0.19 (n = 18) for K85E, and 16.47 ± 0.55 (n = 19) for K85E-E173K. SOCE responses from K85E mutant were significantly lower than those of WT (^∗∗∗, t test). Please see Figure S5B for typical time courses and statistics of current measurements. (C) FRET signals between STIM1 and hOrai1 before and after the addition of 2.5 μM ionomycin. Ionomycin-induced peak ΔE_app: 0.028 ± 0.001 (n = 77) for K85E, 0.033 ± 0.001(n = 84) for K85E-E173K. Both are significantly lower than that of WT (0.073 ± 0.002, n = 73) (^∗∗∗, t test). (D) Confocal imaging results showing typical co-localization of Orai1s with STIM1 after store depletion (scale bar, 10 μm). Please see Figure S5A for complete set of images, and Figure S5B for statistics.

(E and F) (E) In Orai1-3 triple KO cells, the effects of K85E or K85E-E173K mutation on SOCE responses mediated by overexpressed Orai1 and endogenous STIM1 (left panel), constitutive Ca²⁺ entry through overexpressed Orai1-SS (middle panel), or Orai1-S (right panel). Mean constitutive Ca²⁺ entry through overexpressed Orai1-SS: WT = 11.17 ± 0.25 (n = 85), K85E = 6.62 ± 0.45 (^∗∗∗, n = 58), K85E-E173K = 11.74 ± 0.38 (n = 91) (middle panel); mean constitutive Ca²⁺ entry through overexpressed Orai1-S: WT = 1.74 ± 0.13 (n = 75), K85E = 0.02 ± 0.01 (^∗∗∗, n = 62), K85E-E173K = 2.61 ± 0.16 (^∗∗∗, n = 89). (t test against control). (F) Airyscan confocal images of YFP-DSOAR1 co-expressed with Orai1 wild-type or its corresponding mutants in STIM1 and STIM2 double KO cells we recently made (Zheng et al., 2018). Left: typical images (scale bar, 5 μm). Middle: magnified detail of the boxed area shown on the left (scale bar, 1 μm). Statistics of cluster densities: 0.28 ± 0.04/μm² for wild-type, 0.04 ± 0.01/μm² for K85E, and 0.31 ± 0.07/μm² for K85E-E173K. Right: hypothetical clustering of wild-type or mutated Orai1 channels by SOAR1 dimers. For (B), (D), (E), and (F) cells were bathed in 0Ca²⁺ solution containing 1 μM TG for 10 min to deplete ER Ca²⁺ store. 1 μM TG was present throughout the recordings. For current measurements, ER Ca²⁺ store was passively depleted by 20 mM BAPTA included in pipette solution. At least three independent repeats were carried out for each experiment.