4-benzoyl-L-phenylalanine

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.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking 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 (Vrev) 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 Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

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.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking 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 (Vrev) 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 Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

In control experiments, we ensured that an intact plasma membrane expression of the mutants could only be achieved in the presence of the UAA and the expression of the respective tRNA/aaRS pair. In the absence of the latter, only weak background fluorescence was detectable in the cytosol, but not in the plasma membrane (Supplementary Fig. ) in line with abolished UV-mediated Ca2+ influx comparable to untransfected cells (Supplementary Fig. ). Furthermore, examination of the UV-evoked Ca2+ currents of cells co-expressing Orai1 A137Bpa-YFP and Orai1-CFP wild-type as a function of the Orai1 A137Bpa:Orai1 fluorescence intensity ratio showed a tendency for higher maximum currents with increasing ratio and highest UV-induced activation when only Orai1 A137Bpa was expressed (Supplementary Fig. ), indicating efficient UAA incorporation. Moreover, rapid UV-mediated activation occured independent of endogenous STIM and Orai isoforms, as shown in CRISPR/Cas9 STIM1/STIM2 double-knockout (DKO), CRISPR/Cas9 STIM1/Orai1 DKO and CRISPR/Cas9 Orai1/Orai2/Orai3 triple KO (TKO) cells (Supplementary Fig. ), although to slightly lower maximum current levels, likely owing to lower expression levels in the KO compared to wild-type HEK293 cells (Supplementary Fig. ). Moreover, we showed that ER Ca2+ levels detected by LAR-GECO1 in HEK293 cells expressing either Orai1 or Orai1 A137Bpa compared to untransfected cells remained comparable before and after UV light irradiation and subsequent thapsigargin (TG)-induced store-depletion led to a similar decrease in Ca2+ levels (Supplementary Fig. ), thus excluding that UV light-triggered Ca2+ entry leads to Ca2+ overload in the ER.