Tags

Type your tag names separated by a space and hit enter

The Orai1 Store-operated Calcium Channel Functions as a Hexamer.
J Biol Chem. 2016 Dec 09; 291(50):25764-25775.JB

Abstract

Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

Authors+Show Affiliations

From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and zhouyd@psu.edu.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.the Beijing Key Laboratory of Gene Resources and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China.From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and dongill@psu.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27780862

Citation

Cai, Xiangyu, et al. "The Orai1 Store-operated Calcium Channel Functions as a Hexamer." The Journal of Biological Chemistry, vol. 291, no. 50, 2016, pp. 25764-25775.
Cai X, Zhou Y, Nwokonko RM, et al. The Orai1 Store-operated Calcium Channel Functions as a Hexamer. J Biol Chem. 2016;291(50):25764-25775.
Cai, X., Zhou, Y., Nwokonko, R. M., Loktionova, N. A., Wang, X., Xin, P., Trebak, M., Wang, Y., & Gill, D. L. (2016). The Orai1 Store-operated Calcium Channel Functions as a Hexamer. The Journal of Biological Chemistry, 291(50), 25764-25775.
Cai X, et al. The Orai1 Store-operated Calcium Channel Functions as a Hexamer. J Biol Chem. 2016 Dec 9;291(50):25764-25775. PubMed PMID: 27780862.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The Orai1 Store-operated Calcium Channel Functions as a Hexamer. AU - Cai,Xiangyu, AU - Zhou,Yandong, AU - Nwokonko,Robert M, AU - Loktionova,Natalia A, AU - Wang,Xianming, AU - Xin,Ping, AU - Trebak,Mohamed, AU - Wang,Youjun, AU - Gill,Donald L, Y1 - 2016/10/25/ PY - 2016/09/14/received PY - 2016/10/17/revised PY - 2016/10/27/pubmed PY - 2017/5/27/medline PY - 2016/10/27/entrez KW - Ca2+ signaling KW - Crac channels KW - Orai channels KW - Orai1 KW - STIM1 KW - calcium KW - calcium channel KW - cell signaling KW - concatemer KW - ion channel KW - signal transduction KW - store-operated channel KW - stromal interaction molecule 1 (STIM1) SP - 25764 EP - 25775 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 291 IS - 50 N2 - Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/27780862/The_Orai1_Store_operated_Calcium_Channel_Functions_as_a_Hexamer_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=27780862 DB - PRIME DP - Unbound Medicine ER -