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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 1. PM-tethered Orai channel M4x peptides recruit cytosolic SOAR dimers and undergo Leu-specific profiles of FRET interaction.

Figure 1.

(A) Diagram of interacting constructs. LK-CFP-Orai-M4x comprises the Lyn kinase PM tether (LK), cyan fluorescent protein (CFP), and M4x peptide (controls were devoid of M4x); yellow fluorescent protein (YFP)-SOAR dimer is the concatenated dimer of SOAR tagged at the N terminus with YFP.

(B) The M4x sequences of Orai channel subtypes highlighting key hydrophobic residues.

(C) Cell localization images (left) and intensity plots (right) of control LK-CFP with YFP-SOAR dimer co-expressed in HEK-Orai1/2/3TKO cells. (AUs, arbitrary units).

(D–F) Cells as in (C) but co-expressing LK-CFP-O1M4x (containing the Orai1 267-301 M4x peptide) (D), LK-CFP-O2M4x (containing the Orai2 228-254 M4x peptide) (E), or LK-CFP-O3M4x (containing the Orai3 276-295 M4x peptide) (F), each with YFP-SOAR dimer.

(G) E-FRET measurements for the interaction of YFP-SOAR dimer with LK-CFP-Orai1-M4x peptide, containing either wild-type Orai1-M4x (O1-WT); Orai1-M4x with the L273D, L276D, or combined L273D/L276D mutations; or the control (LK-CFP).

(H) E-FRET between YFP-SOAR dimer and LK-CFP-Orai2-M4x peptide, with either WT Orai2-M4x (O2-WT), Orai2-M4x-I234D (O2-I234D), or the control (LK-CFP).

(I) E-FRET between YFP-SOAR dimer and LK-CFP-Orai3-M4x peptide, with either WT Orai3-M4x (O3-WT), Orai3-M4x-L282D (O3-L282D), Orai3-M4x-L285D (O3-L285D), Orai3-M4x-L282D/L285D double mutant (O3-L282D/L285D), or the control (LK-CFP).

(J) Cells as in (C) but co-expressing LK-CFP-O3M4x-L285D mutant with YFP-SOAR dimer. Images and intensity plots are representative of three independently repeated experiments. Scale bars represent 5 μm. One-way ANOVA analysis was performed on E-FRET results (**p < 0.01, ***p < 0.001, ****p < 0.0001). Results are means ± SEM, representative of at least three independent experiments. E-FRET analyses were on cells expressing a narrow range of LK-CFP-Orai-M4x fluorescence to assure accuracy of E-FRET values. The CFP levels are shown in Figure S3.