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Communication between N terminus and loop2 tunes Orai activation.
J Biol Chem. 2018 01 26; 293(4):1271-1285.JB

Abstract

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.

Authors+Show Affiliations

From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.the Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady, Czech Republic.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.the Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady, Czech Republic.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and.the Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady, Czech Republic.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and christoph.romanin@jku.at.From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, and Isabella.derler@jku.at.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29237733

Citation

Fahrner, Marc, et al. "Communication Between N Terminus and Loop2 Tunes Orai Activation." The Journal of Biological Chemistry, vol. 293, no. 4, 2018, pp. 1271-1285.
Fahrner M, Pandey SK, Muik M, et al. Communication between N terminus and loop2 tunes Orai activation. J Biol Chem. 2018;293(4):1271-1285.
Fahrner, M., Pandey, S. K., Muik, M., Traxler, L., Butorac, C., Stadlbauer, M., Zayats, V., Krizova, A., Plenk, P., Frischauf, I., Schindl, R., Gruber, H. J., Hinterdorfer, P., Ettrich, R., Romanin, C., & Derler, I. (2018). Communication between N terminus and loop2 tunes Orai activation. The Journal of Biological Chemistry, 293(4), 1271-1285. https://doi.org/10.1074/jbc.M117.812693
Fahrner M, et al. Communication Between N Terminus and Loop2 Tunes Orai Activation. J Biol Chem. 2018 01 26;293(4):1271-1285. PubMed PMID: 29237733.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Communication between N terminus and loop2 tunes Orai activation. AU - Fahrner,Marc, AU - Pandey,Saurabh K, AU - Muik,Martin, AU - Traxler,Lukas, AU - Butorac,Carmen, AU - Stadlbauer,Michael, AU - Zayats,Vasilina, AU - Krizova,Adéla, AU - Plenk,Peter, AU - Frischauf,Irene, AU - Schindl,Rainer, AU - Gruber,Hermann J, AU - Hinterdorfer,Peter, AU - Ettrich,Rüdiger, AU - Romanin,Christoph, AU - Derler,Isabella, Y1 - 2017/12/13/ PY - 2017/08/16/received PY - 2017/12/12/revised PY - 2017/12/15/pubmed PY - 2018/11/28/medline PY - 2017/12/15/entrez KW - atomic force microscopy (AFM) KW - calcium release-activated calcium channel protein 1 (ORAI1) KW - electrophysiology KW - signal transduction KW - stromal interaction molecule 1 (STIM1) SP - 1271 EP - 1285 JF - The Journal of biological chemistry JO - J Biol Chem VL - 293 IS - 4 N2 - 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/29237733/Communication_between_N_terminus_and_loop2_tunes_Orai_activation_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)39129-8 DB - PRIME DP - Unbound Medicine ER -