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首页> 外文期刊>Nature >The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers
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The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers

机译：CRAC通道由通过刺激诱导的Orai二聚体形成的四聚体组成

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摘要

Ca~(2+)-release-activated Ca~(2+) (CRAC) channels underlie sustained Ca~(2+) signalling in lymphocytes and numerous other cells after Ca~(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca~(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipita-tion and by Forster resonance energy transfer, is involved in the opening of the Ca~(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electro-phoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca~(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photo-bleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca~(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.
机译：Ca〜（2+）释放激活的Ca〜（2+）（CRAC）通道是Ca〜（2+）从内质网（ER）释放后淋巴细胞和许多其他细胞中持续Ca〜（2+）信号的基础。 RNA干扰筛选方法确定了两种蛋白质Stim和Orai，它们共同构成了CRAC通道活性的分子基础。 Stim感觉到ER Ca〜（2+）存储的枯竭，并通过从ER转运到质膜附近的连接处来物理传递此信息，Orai体现了质膜钙通道的孔。通过共免疫沉淀和Forster共振能量转移确定的Stim和Orai之间的紧密相互作用涉及由Orai亚基形成的Ca〜（2+）通道的开放。大多数离子通道是围绕中央通道的成孔亚基的多聚体，它们预先组装在ER中，并以其最终化学计量传输到质膜。在这里，我们通过细胞裂解液和完整细胞中交联后的生化分析，以及通过使用不交联的非变性凝胶电泳显示，Orai在静止条件下主要是质膜中的二聚体。此外，非洲爪蟾卵母细胞中表达的带有绿色荧光蛋白（GFP）标记的Orai的单分子成像显示主要为两步光致漂白，再次与二聚体基础状态一致。相比之下，GFP标记的Orai与Stim的羧基末端作为胞质蛋白共表达以激活Orai通道而不诱导Ca〜（2+）存储耗尽或Orai成簇成点状，主要产生四步光致漂白，与有效Orai通道的四聚体化学计量一致。因此，与Stim的C末端相互作用会诱导Orai二聚体二聚化，形成构成Ca〜（2+）选择性孔的四聚体。这代表了一种新的机制，其中功能性离子通道的组装和激活由相同的触发分子介导。

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