Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

Abstract

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

Figure 1. The STIM-Orai Activating Regions (SOAR) of STIM1 and STIM2 are functionally distinct

Fura-2 ratiometric responses to cytosolic Ca²⁺ in HEK-Orai1 cells transiently expressing YFP-SOAR1 (a) or YFP-SOAR2 (b). SOAR-induced constitutive Ca²⁺ entry was measured after Ca²⁺-free medium was replaced with 1 mM Ca²⁺ followed by 50 µM 2-APB with 1 mM Ca²⁺ (arrows). c, Average peak constitutive Ca²⁺ entry without 2-APB ± SEM (n≥10). d, Average YFP fluorescence intensity (arbitrary fluorescence units; AFU) of cells in (A) and (B) ± SEM (n≥10). e, Expression of YFP-SOAR1, YFP-SOAR2, and Orai1-CFP detected with GFP-antibody, compared to actin expression. f, Cytosolic distribution of YFP-SOAR1 in HEK-Orai1 cells at rest, or 1 min or 5 min after application of 50 µM 2-APB. g, Cellular localization of YFP-SOAR2 under the same conditions as in f. h–k, N-FRET between Orai1-CFP and either YFP-SOAR1 or YFP-SOAR2. h, Mean resting N-FRET for SOAR 1 and SOAR2 (N-FRETrest) ± SEM (n≥6). j, Changes in N-FRET in response to 50 µM 2-APB. k, N-FRET changes relative to resting N-FRET (N-FRET_rest) of response to 50 µM 2-APB shown in (M). Ca²⁺ imaging and FRET responses are means ± SEM of multiple cells (5–17) per field. All results are, typical of at least 3 independent experiments (***p<0.001, unpaired Student’s t-test). Bar = 10 µm.

Figure 2. Structure and sequence of SOAR1 and SOAR2 molecules and construction of SOAR chimeras

a, Cartoon of dimeric human SOAR1 structure showing the four α-helices (Sα1-Sα4) as determined from the crystal structure. b, Alignment of human SOAR1 and SOAR2 proteins showing identical (red), conservatively differing (blue) and non-conservatively differing amino acids (black). The four α-helices are indicated by yellow boxes. Residues involved in inter-dimer interactions are shown, as well as the positive charges that may contribute to STIM-Orai interactions^,,. The α-helical structure of SOAR2 is predicted to be the same as SOAR1 using homology modeling software (Biological Assembly Modeler (BAM) and MolIDE; see Methods and Fig. 9). c, SOAR chimeras used in studies. The Sα1, Sα4, and Sα2 helices in SOAR1 were each individually replaced with the corresponding α-helix from SOAR2. Similarly, the Sα1, Sα4, and Sα2 helices in SOAR2 were each replaced with the corresponding α-helix from SOAR1. The chimeras generated were named according to the four α-helices they contain with α-helices from SOAR1 and SOAR2 color coded in red and green, respectively. For example, replacement of Sα1 in SOAR1 with Sα1 from SOAR2 is named, “SOAR-2111”, the last four digits reflecting the origin (SOAR1 or SOAR2) or each of the four α-helices. We did not exchange Sα3 helices since they are the same; thus, the α-helices in SOAR-1122 are the same as those in SOAR-1112, and those in SOAR-2211 are the same as in SOAR-2221. The Sα2 helices differ by only one amino acid; therefore, SOAR-1211 is the same as the point mutation, SOAR1-F394L, and SOAR-2122 is the same as the point mutation, SOAR2-L485F. All chimeras were N-terminally tagged with YFP.

Figure 3. Removal of the C-terminal variable regions from the STIM1ct and STIM2ct EA-mutants does not alter their distinct Orai1-activation and Orai1-binding properties

The STIM1 and STIM2 C-terminal constructs used in Supplementary Fig. S2 were each truncated to remove their C-terminal variable regions to give the STIM1ctΔV-4EA-YFP and STIM2ctΔV-3EA-YFP, as shown in Supplementary Fig. S1. Expressed in HEK-Orai1 cells, the Ca²⁺ responses for STIM1ctΔV-4EA-YFP (a) or STIM2ctΔV-3EA-YFP (b) are shown after 1 mM Ca²⁺ or 50 µM 2-APB addition. Cells expressed almost equal levels of YFP fluorescence (c; means ± SEM), and Western analysis revealed similar protein levels (d) detected with GFP antibody. Cytosolic distribution of STIM1ctΔV-4EA-YFP (e) or STIM2ctΔV-3EA-YFP (f) at rest. g, Resting N-FRET levels between Orai1-CFP and STIM1ctΔV-4EA-YFP (red) or STIM2ctΔV-3EA-YFP (green) (p<0.001 (n≥6) and changes in N-FRET following 50 µM 2-APB application (h). Ca²⁺ imaging and FRET responses are means ± SEM of multiple cells (7–26) per field. Results are typical of at least 3 independent experiments (***p<0.001, unpaired Student’s t-test). Bar = 10 µm.

Figure 4. The Sα1 and Sα2 helices of SOAR1 and SOAR2 crucially determine differences in binding and activation of the Orai1 channel

Comparative function of YFP-SOAR1 (a), YFP-SOAR2 (b), the Sα1-exchanged chimeras, YFP-SOAR-2111 (c) and YFP-SOAR-1222 (d), and the Sα2-exchanged chimeras, YFP-SOAR-1211 (e) and YFP-SOAR-2122 (f), expressed in HEK-Orai1 cells. Fura-2 measurements (upper row) were in Ca²⁺-free medium with addition of 1 mM extracellular Ca²⁺ and 50 µM 2-APB (arrows). FRET measurements (middle row) were with 1 mM Ca²⁺ with 50 µM 2-APB added (arrows). Deconvolved images of YFP-SOAR distribution (lower row) were with 1 mM Ca²⁺. g, Mean of peak Ca²⁺ entry without 2-APB ± SEM (n≥6). h, Mean of resting N-FRET ± SEM (n≥5). *** differences from SOAR1, p<0.001 (unpaired Student’s t-test). Ca²⁺ imaging and FRET results are means ± SEM of multiple cells (5–17) per field, typical of at least 3 independent experiments. Bar = 10 µm.

Figure 5. The Sα4 helices of SOAR1 and SOAR2 do not contribute to their distinct binding properties or activation of the Orai1 channel

Comparative function of YFP-SOAR1 (a), YFP-SOAR2 (b), and the Sα4-exchanged chimeras, YFP-SOAR-1112 (c) and YFP-SOAR-2221 (d), expressed in HEK-Orai1 cells. Fura-2 measurements (upper row) were in Ca²⁺-free medium with addition of 1 mM extracellular Ca²⁺ and 50 µM 2-APB (arrows). FRET measurements (middle row) were with 1 mM Ca²⁺ with 50 µM 2-APB added (arrows). Deconvolved images of YFP-SOAR distribution (lower row) were with 1 mM Ca²⁺. e, Mean of peak Ca²⁺ entry without 2-APB ± SEM (n≥7). f, Mean of resting N-FRET ± SEM (n≥4). *** differences from SOAR1, p<0.001, unpaired Student’s t test. Ca²⁺ imaging and FRET responses are means ± SEM of multiple cells (5–20) per field and all results are typical of at least 3 independent experiments. Bar = 10 µm.

Figure 6. The F394 residue within full length STIM1 functions as a pivotal locus for both binding to and gating of Orai1 channels

a, Fura-2 Ca²⁺ measurements undertaken on HEK-Orai1 cells expressing wildtype STIM1-YFP (black), or STIM1 mutants F394L-YFP (green), F394A-YFP (blue), or F394H-YFP (red). Cells in Ca²⁺-free medium were treated with 2.5 µM ionomycin, 1 mM Ca²⁺, and 50 µM 2-APB (arrows). b, Average ± SEM YFP fluorescence intensity of cells in a. c, Measurements as in a but without ionomycin addition. d–g, Whole cell ICRAC measurements on HEK-Orai1 cells transfected with wildtype STIM1-YFP (d), or the STIM1 mutants, F394L-YFP (e), F394A-YFP (f), or F394H-YFP (g); currents were induced by passive store-depletion with 20mM EGTA in the pipette, and subsequent addition of 50µM 2-APB in the bath. Typical traces are shown of whole cell currents at −100mV. h,i, I-V relationship from cells shown in d–g either at peak store-operated current (orange arrow) or peak 2-APB induced current (purple arrow). j, N-FRET change between Orai1-CFP and STIM1-YFP using the same mutants expressed within HEK-Orai1 cells, as in a. Additions of 2.5 µM ionomycin and 50 µM 2-APB in Ca²⁺-free medium were as shown. k, N-FRET measurements as in j but without ionomycin addition. l–n, Cytosolic distribution of Orai1-CFP (red) and STIM1-F394H-YFP (green) expressed in HEK-Orai1 cells. Images are of a single cell before (l), and after 5 min treatment with 2.5 µM ionomycin (m), and a further 5 min with 50 µM 2-APB (n). Detailed quantitative analysis and statistics on Ca²⁺ entry, FRET, peak ICRAC, and Orai1/STIM1transocation into puncta are given in Fig. 7. Ca²⁺ imaging and FRET responses are means ± SEM of multiple cells (8–33) per field and results are all typical of at least 3 independent experiments. Bar = 10 µm.

Figure 7. Summary data for the effects of STIM1-F394 mutants on SOCE, Orai1 N-FRET, I_CRAC, and punctal changes, shown in Fig. 6

a, Comparison of peak ionomycin-induced SOCE in HEK-Orai1 cells transfected with STIM1-YFP, STIM1-F394L-YFP, STIM1-F394A-YFP, or STIM1-F394H-YFP as shown in Fig. 4A (***, p<0.001, unpaired Student’s t test, n=6). b, Ionomycin-induced peak ΔN-FRET/N-FRET_rest (%) with the same STIM1-YFP constructs used in a, as shown in Fig. 6j (***, p<0.001, unpaired Student’s t test, n≥4). c, Peak ICRAC activity in HEK-Orai1 cells transfected with the same STIM1-YFP constructs used in (a) in response to passive store-depletion, as shown in Fig. 6d–g. d, Additional ICRAC activity induced by 2-APB in the same cells as shown in (c) (** p<0.01;*** p<0.001; unpaired Student’s t test, WT n=6; F394L n=4; F394A n=4; F394H n=6). e,f, Quantitative analysis of ionomycin-induced STIM1-F394H-YFP and Orai1 movement into puncta (red; data in Fig. 6l,m) compared to movement of wildtype-STIM-YFP in HEK-Orai cells (black). For STIM1 (e), the Δ(YFP_puncta/YFP_control)% values for wildtype STIM1-YFP and STIM1-F394H-YFP are similar. For Orai1 (f), the Δ(CFP_puncta/CFP_control)% for cells expressing STIM1-F394H-YFP is substantially reduced compared to cells expressing wildtype STIM1. Thus, the STIM1-F394H mutant has substantially reduced Orai-binding activity (means ± SEM, ***, p<0.001; unpaired Student’s t test).

Figure 8

The F394 residue critically determines binding between STIM1 and Orai1. Immunoprecipitation experiments were undertaken in HEK cells stably expressing either Orai1-CFP and STIM1-YFP (S1-WT), or Orai1-CFP and STIM1-F394H-YFP (S1-F394H). Cells were treated as indicated before lysis. Whole cell lysates (left) and STIM1 antibody immunoprecipiates (right) are shown. Detection utilized either STIM1 antibody or GFP antibody to detect Orai1-CFP. Whereas STIM1-WT pulls down Orai1 (right, lane 1), the S1-F394H mutation is almost devoid of Orai1 interaction (right, lane 2). 2-APB enhances the interaction between S1-F394H and Orai1 after ionomycin treatment (right, lane 3). Original membrane scans are given in Supplementary Fig. S3.

Figure 9. Structural configuration of the distinct Orai-coupling domains of STIM1 and STIM2

a. Structural comparison between SOAR1 and SOAR2 dimers. The structure of SOAR1 is derived from the crystal structure (red). Homology modeling of residues in SOAR2 using the Biological Assembly Modeler (BAM) and MolIDE software (green) reveal the backbone ribbons for SOAR1 and SOAR2 superimpose almost exactly throughout the two molecules. The model shows only the sidechains present in the apical Sα1/Sα2 regions that differ between SOAR1 and SOAR2 (these are identified in b). SOAR1 sidechains are shown as orange, SOAR2 as light blue. b, Detail from a of the two apical domains of SOAR1 and SOAR2 identifying the eight amino acids that differ in this region – seven residues in Sα1 and one in Sα2 are shown. The four non-conservative changes (K371/M462, L374/A465, G379/E470, N388/S479; yellow shaded) may contribute to the differential Orai-binding of STIM1 and STIM2. The F394/L485 substitution in Sα2 is crucial for gating differences between STIM1 and STIM2. c–f, Structural modeling to show preferred rotamers of STIM1-F394 substituted sidechains: c, phenylalanine (in STIM1) functions as a strong Orai; d, leucine (in STIM2) functions as a partial agonist; e, alanine, functions as a weak agonist; f, histidine, functions as a non-agonist.