The Orai1 Store-operated Calcium Channel Functions as a Hexamer
The Orai1 Store-operated Calcium Channel Functions as a Hexamer
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DOI：
10.1074/jbc.m116.758813
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发表时间：
2016-12-09
影响因子：
4.8
通讯作者：
Gill, Donald L.
中科院分区：
文献类型：
--
作者：
Cai, Xiangyu;Zhou, Yandong;Gill, Donald L.
ORAI通道参与调节多种细胞转录的钙离子信号，并参与多种免疫学和炎症性疾病。尽管Orai通道的基本亚基结构至关重要，但围绕它们的争议仍然存在，几项生化和生物物理研究表明，Orai通道是四聚体结构，而结晶学证据表明是六聚体。我们系统地研究了功能Orai1通道的亚基结构，产生了一系列在CRISPR衍生的ORAI1基因敲除HEK细胞中表达的tdTomato标记的串联Orai1通道结构(二聚体到六聚体)，稳定表达STIM1-YFP。表面生物素化反应表明，全长串联体为表面膜表达。出乎意料的是，Orai1二聚体、三聚体、四聚体、五聚体和六聚体都介导了类似的、实质性的由存储操作的钙离子进入。此外，每个Orai1串联体介导的钙电流具有与表达的Orai1单体几乎相同的内向整流和翻转电位。在Orai1四聚体中，用Orai1E106A“孔不活跃”亚基进行的亚基特异性替换揭示了功能通道只利用来自四聚体的N-末端二聚体。相反，Orai1E106A在Orai1六聚体中的替换建立了所有亚基都可以促进通道形成的事实，表明了一种六面体通道构型。关键的钙离子选择性过滤器-形成Glu-106残基可能介导Orai1通道围绕孔内中央钙离子组装。因此，Orai1六聚体中的多个E106A替换可能促进一种替代的“三聚体-二聚体”通道构型，其中C-末端E106A亚单位被排除在六聚体核心之外。我们的结果强烈反对Orai1通道的四聚体构型，并表明Orai1通道起到六聚体的作用。
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.