title: The Orai1 Store-operated Calcium Channel Functions as a Hexamer.
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: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Xiangyu
authors: X
authors: Cai
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and zhouyd@psu.edu.
authors: Yandong
authors: Y
authors: Zhou
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Robert M
authors: RM
authors: Nwokonko
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Natalia A
authors: NA
authors: Loktionova
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Xianming
authors: X
authors: Wang
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Ping
authors: P
authors: Xin
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and.
authors: Mohamed
authors: M
authors: Trebak
authors: the Beijing Key Laboratory of Gene Resources and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors: Youjun
authors: Y
authors: Wang
authors: From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and dongill@psu.edu.
authors: Donald L
authors: DL
authors: Gill
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D000071740:ORAI1 Protein / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*
chemical_list: D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D002118:Calcium
keywords: Ca2+ signaling; Crac channels; Orai channels; Orai1; STIM1; calcium; calcium channel; cell signaling; concatemer; ion channel; signal transduction; store-operated channel; stromal interaction molecule 1 (STIM1)