Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1

Abstract

Ca²⁺ release-activated Ca²⁺ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca²⁺-sensor STIM1 in the ER membrane and the Ca²⁺ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca²⁺ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca²⁺ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Fig. 1. Site-specific incorporation of Azi and Bpa in Orai1.

a Chemical structures of Azi and Bpa. b Scheme of the TM domains of an Orai1 subunit. Stars indicate sites for UAA incorporation. c Principle of UAA incorporation in mammalian cells. A gene encoding a protein of interest (here Orai1) containing an Amber stop codon (TAG) inserted at the desired site is co-transfected with a bioorthogonal tRNA (blue)/aminoacyl synthetase (RS) pair (yellow) that does not crosstalk with endogenous pairs (black/ pink). Cells are incubated with the UAA supplemented in the medium. Within the host cell, the suppressor tRNA, aminoacylated with the UAA by the bioorthogonal RS, recognizes the Amber stop codon on the mRNA (UAG codon, red) at the ribosome to insert the UAA into the nascent amino acid chain. Different light-sensitive Orai1 mutants are generated carrying the photocrosslinking UAAs at the desired sites. d The table summarizes the effects on Ca²⁺ influx of all screened Orai1 mutants containing Azi or Bpa at a position in TM2-4 after UAA incorporation and after UV light (365 nm) irradiation. A cross indicates no activity (below threshold line) after UAA insertion. Small (no significant change in activity (n.s.), but above threshold line (see Supplementary Fig. 1)) or large (significant change in activity and above threshold line (*)) check marks indicate constitutive activity upon switching from a 0 mM to a 2 mM Ca²⁺-containing solution. UV illumination (10 s) can lead to activation which is highlighted by small (no significant change in activity before versus after UV light, but above threshold line (n.s.)) or large (significant change in activity before after after UV light and above threshold (*)), purple upward-facing triangles. UV-induced deactivation is illustrated by small (reducted activity, but not significant (n.s.)) or large (significantly reduced activity (*)), yellow downward-facing triangles. Mutants with no change in activity upon UV illumination are marked with a red slash. Accordingly, UV-induced effects (indicated by the color gradient) are represented in the top- and side-view cartoons of Orai1 in the respective colors. Corresponding data are shown in Supplementary Fig. 1 and provided as a Source Data file.

Fig. 2. Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.

Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca²⁺ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca²⁺ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca²⁺ solution followed by a 2 mM Ca²⁺ solution. Under 2 mM Ca²⁺ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca²⁺ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca²⁺ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (V_rev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that V_rev are not significantly different. Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 3. STIM1 modulates the activity of photocrosslinking UAA-containing Orai1 mutants.

Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the presence of STIM1. Stars indicate sites for UAA incorporation. a–c Time courses of Ca²⁺ current densities after whole-cell break-in of the above mentioned Orai1 mutants co-expressed with STIM1. UV light was applied for 15 s leading to UV-induced Orai1 activation after reaching maximum STIM1-mediated Orai1 mutant activation. Time courses in light gray show the respective Orai1 mutants in the absence of STIM1. d–f Corresponding I/V relationships were taken from indicated time points (black and colored circles, respectively) in (a–c). Inlet represents the reversal potential (V_rev) of STIM1-activated Orai1 channel currents versus STIM1-activated light-sensitive Orai1 mutants currents before and after application of UV light. Single values are indicated in gray. g–i Time courses of current densities after whole-cell break-in of the above mentioned UAA-containing Orai1 mutants co-expressed with STIM1. UV light was applied for 15 s at time segments indicated by colored bars, either before or after passive store-depletion mediated activation. j Confocal fluorescence microscopy images of representative cells before and after treatment with 1 μM thapsigargin (TG) showing STIM1-CFP, Orai1-YFP or Orai1 A137Bpa-YFP as well as their overlay. Images were recorded after the application of 10 s UV light. White bars indicate 5 µm. Time courses of FRET (E_app) monitoring the interaction of STIM1 with wild-type Orai1 or Orai1 A137Bpa when switching from a 2 mM Ca²⁺-containing solution to a 0 mM Ca²⁺/1μM TG solution inducing STIM1/Orai1 interaction without (k) or after (l) application of 10 s UV light. m Corresponding bar diagram to (k) and (l) comparing FRET of STIM1/wild-type Orai1 and STIM1/Orai1 A137Bpa before and after treatment with 1 μM TG as well as with and without application of UV light (Welch-ANOVA for (m) + Fig. 4c: F(7;20,37)=86,17, p = 1.53*10⁻¹³). Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05). Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 4. STIM1 is not required for UV-mediated activation of photocrosslinking UAA-containing Orai1 mutants.

a Time courses of current densities after whole-cell break-in of Orai1 A137Bpa or Orai1 A137Bpa L273D co-expressed with either STIM1 or STIM1 L373S A376S. UV light is applied for 15 s at t = 250 s, the time point after maximum STIM1-mediated Orai1 mutant activation indicating completed store-depletion. b Bar diagram summarizes UV-induced maximum currents measured for Orai1 A137Bpa or Orai1 A137Bpa L273D in the absence of STIM1 or the presence of STIM1, STIM1 L373S or STIM1 L373S A376S. Single values are indicated in gray (one-way ANOVA for (b): F(7;99) = 2,15, p = 0.046). c Bar diagram of FRET (E_app) comparing STIM1 or STIM1 L373S A376S co-expressed with Orai1 A137Bpa or Orai1 A137Bpa L273D before and after treatment with 1 μM TG as well as with and without application of UV light (Welch-ANOVA for (c) + Fig. 3m: F(7;20,37) = 86.17, p = 1.53*10⁻¹³). d Confocal fluorescence microscopy images of representative cells before and after treatment with 1 μM TG showing STIM1-CFP, Orai1 A137Bpa-YFP or Orai1 A137Bpa L273D-YFP as well as an overlay of both after application of 10 s UV light. White bars indicate 5 µm. Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05). Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 5. Photocrosslinking induced Orai1 activation delays the return to the resting state and leaves STIM1 coupling unaffected.

a Ca²⁺ imaging measurements showing intracellular Ca²⁺ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with Orai1 A137Bpa either without or with STIM1, while switching from a 0 mM Ca²⁺ to a 0 mM Ca²⁺/100 µM CCH/50 µM BHQ and finally to a 2 mM Ca²⁺-containing solution. Blue trace shows Orai1 A137Bpa currents without 100 µM CCH/50 µM BHQ application. UV light was applied for 10 s to cells expressing Orai1 A137Bpa or STIM1 + Orai1 A137Bpa and compared to STIM1 + Orai1 A137Bpa evoked Ca²⁺ levels in the absence of UV light. b Summarizing bar diagram comparing Ca²⁺ levels in (a) and of STIM1 + wild-type Orai1 at maximal levels and at t = 600 s (one-way ANOVA for (b): F(9;160,73) = 9,79, p = 6,96*10⁻¹²). c Time courses of FRET (E_app) values monitoring the interaction of STIM1 with wild-type Orai1 or Orai1 A137Bpa when switching from a 2 mM Ca²⁺ solution to a 0 mM Ca²⁺/10 µM BHQ solution and subsequently to a 2 mM Ca²⁺ solution. UV light irradiation was applied for 10 s subsequent to store-depletion in 2 mM Ca²⁺ solution. d Summarizing bar diagram of FRET (E_app) values corresponding to (c) at indicated time points (t = 0 (Start), 6 (Max), 12 min (End)). Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05). Detailed statistic values are shown in detail in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 6. GoF mutations in Orai1 TM domains, but not Orai1 nexus mutation, interfere with UV-mediated activation of photocrosslinking UAA-containing Orai1 mutants.

Graphical illustration on the left-hand side represents photocrosslinking UAA-containing Orai1 mutants, exemplarily shown for Orai1 A137Bpa (star), combined with one of the following GoF mutations: V102A, H134A, V181K, P245L and ₂₆₁ANSGA₂₆₅ (black line). a Bar diagram summarizes currents measured before and after application of UV light for the above mentioned photocrosslinking UAA- and GoF-containing Orai1 double mutants (Welch-ANOVA for Orai1 A137Bpa mutants: F(11;21,41) = 7, p = 7.5*10⁻⁵; for Orai1 L174Bpa mutants F(11;27,79) = 16,48, p = 1.96*10⁻⁹; for Orai1 A254Azi mutants F(11;32,59) = 15,15, p = 6.72*10⁻¹⁰). b Bar diagram summarizes normalized R-GECO1.2 intensities measured before and after application of UV light corresponding to (a) (Welch-ANOVA for (b): F(35;326,09)=27,11, p = 0). Time course of current densities after whole-cell break-in of Orai1 A137Bpa ₂₆₁ANSGA₂₆₅ compared to Orai1 A137Bpa (c), Orai1 L174Bpa ₂₆₁ANSGA₂₆₅ compared to Orai1 L174Bpa (d) and Orai1 A254Azi ₂₆₁ANSGA₂₆₅ compared to Orai1 A254Azi (e). UV light was applied for 15 s. Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05). Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 7. Light-sensitive Orai1 mutants require an intact channel geometry.

Graphical illustration on the left-hand side represent photocrosslinking UAA-containing Orai1 mutants, exemplarily shown for Orai1 A137Bpa (star), combined with the pore mutation E106Q (stop sign), various N-terminal truncations (scissor) or cytosolic extended TM region (CETR)—LOF (stop sign) mutations. Time course of current densities after whole-cell break-in comparing Orai1 A137Bpa and Orai1 A137Bpa E106Q (a), Orai1 L174Bpa and Orai1 L174Bpa E106Q (b) and Orai1 A254Azi and Orai1 A254Azi E106Q (c). Time course of current densities after whole-cell break-in comparing the light-sensitive Orai1 mutants (Orai1 A137Bpa (d), Orai1 L174Bpa (e) and Orai1 A254Azi (f)) with different corresponding N-terminally truncated mutants (Orai1 A137Bpa Δ1-47/64/72/78; Orai1 L174Bpa Δ1-47/64/72/78; Orai1 A254Azi Δ1-47/64/72/78). Time course of current densities after whole-cell break-in comparing the light-sensitive Orai1 mutants (Orai1 A137Bpa (g), Orai1 L174Bpa (h) and Orai1 A254Azi (i)) with different corresponding mutants containing different CETR-LoF mutations (K85E, E149K and L174D). In all cases (a-i) UV light is applied for 15 s. Data represent mean values ± SEM of indicated number (n) of experiments. Source data are provided as a Source Data file.

Fig. 8. UV light-activated Orai1 A137Bpa currents match CRAC channel hallmarks best.

a Time course of current densities after whole-cell break-in comparing Orai1 A137Bpa with wild-type STIM1/Orai1, while repeatedly switching from 10 mM Ca²⁺- (I_Ca2+) to a DVF Na⁺- (I_Na+-DVF) containing solution. 15 s UV pulse was only applied to Orai1 A137Bpa. b Bar diagram summarizing the ratio of currents (I_Na+-DVF/I_Ca2+) for conditions shown in (a) and for Orai1 A137Bpa with STIM1 and Orai1 L174Bpa and Orai1 A254Azi in the absence and presence of STIM1 with and without UV light (15 s). Single values are indicated in gray. An increase in the I_Na+-DVF was measured when the threshold marked in blue was exceeded (Welch-ANOVA for (b): F(9;38,52)=62,85, p = 0). c Time course showing normalized currents of Orai1 A137Bpa compared to wild-type CRAC channel (STIM1 + Orai1; WT) obtained upon application of a voltage step to −70 mV from a holding potential of 0 mV using 20 mM EGTA in the pipette. d Bar diagram summarizing normalized currents at t = 250 ms from data depicted in (c) and for other conditions and mutants in analogy to (b). Single values are indicated in gray (Welch-ANOVA for (d): F(9;23,63) = 12,79, p = 3,54*10⁻⁷). e Time course showing normalized currents in analogy to (c) using 20 mM BAPTA in the pipette. f Bar diagram summarizing normalized currents at t = 250 ms from data depicted in (c) and (e) using either 20 mM EGTA or 20 mM BAPTA in the pipette and 10 mM Ca²⁺ or Na⁺-containing DVF solution at the extracellular side (Welch-ANOVA for (f): F(7;15,82)=13,41, p = 1,27*10⁻⁵). g Time courses of Ca²⁺ current-densities after whole-cell break-in of Orai1 A137Bpa, Orai1 L174Bpa and Orai1 A254Azi exposed to UV-light (15 s) compared to STIM1/Orai1 currents activated by passive store-depletion. After maximal activation the Orai1 channel blocker CM-4620 (10 µM) was applied. h Summarizing bar diagram comparing current-densities in (g) at maximal levels and after application of CM-4620 (t = 320 s; Welch-ANOVA for (h): F(7;12,19)=39,98, p = 2,2*10⁻⁷). Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05). Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 9. Photocrosslinking of light-sensitive Orai1 mutants trigger Ca²⁺-dependent downstream signaling.

Time course of current densities after whole-cell break-in comparing Orai1 A137Bpa and Orai1 A254Azi in RBL-2H3 cells (a) and Jurkat TIB-152 cells (d). b, e Corresponding bar diagram to (a) and (d), respectively. Values taken from time points 25 s and 125 s (a) and 15 s and 75 s (d), respectively. c, f Corresponding I/V relationships were taken at 125 s in (a) and 75 s in (d). Inlet represents reversal potential (V_rev) of light-sensitive Orai1 mutant currents. Time course of NFAT translocation into the nucleus (normalized ratio NFAT (core/cytosol (cyt)) indicating the fluorescence ratio nucleus:cytosol) of Orai1 A137Bpa with and without (only in (g)) application of UV light compared to wild-type Orai1 in HEK293 (g) or RBL-2H3 (j) cells. NFAT translocation was monitored initially in 0 mM Ca²⁺ solution, followed by 2 mM Ca²⁺ solution after 10 min together with the application of UV light (10 s). h, k Bar diagram summarizing the extent of NFAT translocation for wild-type Orai1, Orai1 A137Bpa, and additionally Orai1 L174Bpa and Orai1 A254Azi with (h) & (k) and without application of UV light (only in (h)) and thapsigargin-activated (+TG) Orai1 expressing cells (only in (k)). Paired bars show the ratio under resting (black, t = 3 min) and activated (colored, t = 35 min) conditions. i and l Fluorescence images of CFP-labeled NFAT of representative cells corresponding to (g), (h), (j) and (k) before and after application of 10 s UV light. White bars indicate 5 µm. Data represent mean values ± SEM of indicated number (n) of experiments. *Significant differences (p < 0.05) tested by Mann–Whitney test. Detailed statistic values are shown in Supplementary Table 3. Source data are provided as a Source Data file.

Fig. 10. Photocrosslinking-induced Orai1 activation leads to CRAC channel-like Ca²⁺ influx suitable to trigger nuclear factor of activated T-cells (NFAT) translocation.

(left) Closed Orai1 channel (represented by two subunits containing four transmembrane (TM) domains) which has a photocrosslinking UAA (star) in one of the TM domains incorporated. (right) Upon application of UV light, photocrosslinking triggers a local conformational change, which is transferred as a global structural change to the entire channel complex and triggers pore opening. Subsequent Ca²⁺ influx leads to the activation of downstream signaling cascades such as the nuclear translocation of NFAT.