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. Author manuscript; available in PMC: 2021 Sep 24.
Published in final edited form as: Cell Rep. 2021 Jun 29;35(13):109322. doi: 10.1016/j.celrep.2021.109322

Figure 5. The Orai3 M4x peptide is a powerful SOCE blocker and defines the physiological role of Orai channels in Ca2+ oscillation generation and downstream NFAT1 activation.

Figure 5.

(A) Endogenous SOCE in WT HEK293 cells transiently expressing LK-CFP-O3M4x (red), LK-CFP-O1M4x (green), or LK-CFP control (black).

(B) SOCE as in (A) in cells transiently expressing LK-CFP-O3M4x (red), LK-CFP-O3M4x-L285D (blue), or LK-CFP control (black).

(C) Summary statistics of endogenous SOCE responses shown in (A) and (B).

(D) Receptor-activated Ca2+ responses induced by 100 μM carbachol (CCh) measured in fura-2-loaded WT HEK293 cells transiently expressing either LK-CFP-O3M4x (red), LK-CFP-O3M4x-L285D mutant (blue), or LK-CFP control (black).

(E–G) Representative single-cell Ca2+ responses to CCh from each of the traces shown in (D) and the fraction of oscillating (OSC) and non-oscillating (NO) cells unde reach condition. Cells were transfected with LK-CFP(77.5% OSC, 22.5 NO; 44 cells) (E), LK-O3M4x-WT (19.7% OSC, 80.3% NO; 71 cells) (F), and LK-CFP-O3M4x-L285D (64.4% OSC, 35.6 NO; 59 cells) (G).

(H–J) Time-dependent nuclear localization of NFAT1 in WT HEK293 cells transiently expressing NFAT1-GFP together with either LK-CFP control (H), LK-CFP-O3M4x (I), or LK-CFP-O3M4x-L285D mutant (J). Images of CFP, CFP/GFP, and GFP were taken before CCh (0 min) and at 3, 5, 10, and 15min after 100 μM CCh addition. Fluorescence of LK-CFP constructs and NFAT1-GFP are shown in red and green, respectively.

(K) Summary of NFAT1-GFP translocation imaged in cells co-expressing LK-CFP (black), LK-CFP-O3M4x-WT (red), and LK-CFP-O3M4x-L285D (blue). Nuclear/cytosolic GFP-NFAT1 intensity ratios were measured in imaged cells treated as in (H)—(J) by using images taken before and after CCh addition, at the times shown. Oscillating cells were defined as having >4 peaks/10 min after initial Ca2+ release (400 s), with each peak at >0.03 F340/F380. One-way ANOVA analyses were undertaken on results in (C). Ca2+ entry results shown are means ± SEM (****p < 0.0001). All results shown are representative of at least three independent experiments. Scale bars represent 10 μm.