Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca2+ concentrations. The primary source of Ca2+ entry into the cell is mediated by the Ca2+ release-activated Ca2+ (CRAC) channel. Its action is stimulated in response to internal Ca2+ store depletion. The fundamental constituents of CRAC channels are the Ca2+ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca2+-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1–Orai1 machinery.

Graphical AbstractOrai1 and Orai3 channel activation depends in an isoform-specific manner on two non-conserved residues in TM3 (Orai1: V181, L185, Orai3: A156, F160). Mutation of these residues to alanine leads in the absence of STIM1 to small constitutive activity of the respective Orai1 mutants, however, to huge constitutive currents of the respective Orai3 mutants. Overall, two non-conserved residues in TM3 control the maintenance of the closed state as well as an opening permissive conformation of Orai channels in an isoform-specific manner.

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.