Communication between N terminus and loop2 tunes Orai activation

• Published in: The Journal of biological chemistry Vol. 293; no. 4; pp. 1271 - 1285
• Main Authors: Fahrner, Marc, Pandey, Saurabh K., Muik, Martin, Traxler, Lukas, Butorac, Carmen, Stadlbauer, Michael, Zayats, Vasilina, Krizova, Adéla, Plenk, Peter, Frischauf, Irene, Schindl, Rainer, Gruber, Hermann J., Hinterdorfer, Peter, Ettrich, Rüdiger, Romanin, Christoph, Derler, Isabella
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-01-2018; American Society for Biochemistry and Molecular Biology
• Subjects: atomic force microscopy (AFM); Calcium Channels - chemistry; Calcium Channels - genetics; Calcium Channels - metabolism; calcium release-activated calcium channel protein 1 (ORAI1); electrophysiology; HEK293 Cells; Humans; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; ORAI1 Protein - chemistry; ORAI1 Protein - genetics; ORAI1 Protein - metabolism; Protein Domains; Protein Structure, Secondary; Signal Transduction; stromal interaction molecule 1 (STIM1); Stromal Interaction Molecule 1 - chemistry; Stromal Interaction Molecule 1 - genetics; Stromal Interaction Molecule 1 - metabolism; calcium release-activated calcium channel protein 1 (ORAI1); stromal interaction molecule 1 (STIM1); atomic force microscopy (AFM); electrophysiology; signal transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.812693

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73–91; Orai3 amino acids 48–65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.