Published January 19, 2023 | Version v1
Journal article Open

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

  • 1. Institute of Biophysics, JKU Life Science Center, Johannes Kepler University Linz, A-4020 Linz, Austria
  • 2. Division of Medical Physics and Biophysics, Gottfried Schatz Research Center, Medical University of Graz, A-8010 Graz, Austria

Description

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. 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 reveals 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.

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