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

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. The Ca2+ ion channel Orai1 is crucial in immune cells. Here, the authors applied genetic code expansion to transfer light-sensitivity to the Orai1 channel and achieved precise control over its function.

Automated summary

CRAC channels are essential for the immune system and various other human body functions. The authors used genetic code expansion to introduce light-sensitivity to the Orai1 channel, allowing precise control over its function. UV light exposure triggered different effects depending on the specific unnatural amino acid incorporated into Orai1, with photoactivation at site A137 using Bpa resulting in Ca2+ currents that closely resemble CRAC channels and can activate downstream signaling pathways without the need for STIM1.