Institute of Biophysics, JKU Life Science Center, Johannes Kepler University Linz, A-4020, Linz, Austria.
Division of Medical Physics and Biophysics, Gottfried Schatz Research Center, Medical University of Graz, A-8010, Graz, Austria.
Nat Commun. 2023 Mar 8;14(1):1286. doi: 10.1038/s41467-023-36458-4.
Ca release-activated Ca (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca-sensor STIM1 in the ER membrane and the Ca 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 Ca 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 Ca 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.
钙释放激活钙 (CRAC) 通道对于免疫系统和人体的各种其他功能至关重要，由两个跨膜 (TM) 蛋白组成，内质网膜中的钙传感器 STIM1 和质膜中的钙离子通道 Orai1。在这里，我们在哺乳动物细胞系中采用遗传密码扩展技术，将光交联非天然氨基酸 (UAA)、对苯甲酰基-L-苯丙氨酸 (Bpa) 和对叠氮基-L-苯丙氨酸 (Azi) 掺入 Orai1 的 TM 结构域的不同位点。使用钙成像和电生理学对各自含有 UAA 的 Orai1 突变体进行表征，结果表明，根据 UAA 及其掺入的位置，紫外光照射会引发一系列不同的效应。特别是，在 Orai1 中使用 Bpa 对 A137 进行光激活可激活钙电流，这些钙电流与 CRAC 通道的生物物理特性最佳匹配，并且能够触发下游信号通路，如激活 T 细胞的核因子 (NFAT) 进入细胞核，而无需生理激活剂 STIM1。
