STIM1 在体外门控钙库操作钙通道 ORAI1。
STIM1 gates the store-operated calcium channel ORAI1 in vitro.
机构信息
Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, Massachusetts, USA.
出版信息
Nat Struct Mol Biol. 2010 Jan;17(1):112-6. doi: 10.1038/nsmb.1724. Epub 2009 Dec 27.
Abstract
Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.
摘要
哺乳动物 T 细胞和肥大细胞中的储存操作的 Ca(2+)内流通过质膜 Ca(2+)释放激活的 Ca(2+)（CRAC）通道，依赖于传感器蛋白基质相互作用分子 1（STIM1）和通道亚基 ORAI1。为了在体外研究 STIM1-ORAI1 信号，我们在酵母 Saccharomyces cerevisiae 的 sec6-4 菌株中表达了人 ORAI1，并从高尔基体区室分离出带有 ORAI1 的密封膜囊泡到质膜。我们通过体外 Ca(2+)通量测定表明，细菌表达的重组 STIM1 打开野生型 ORAI1 通道，但不打开由 ORAI1 孔突变体 E106Q 或 ORAI1 严重联合免疫缺陷（SCID）突变体 R91W 组装的通道。这些实验表明，在没有其他人类 ORAI1 通道复合物蛋白的情况下，STIM1-ORAI1 相互作用足以门控重组人 ORAI1 通道，并且为进一步的生化和生物物理剖析 ORAI1 通道门控奠定了基础。
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Increased hydrophobicity at the N terminus/membrane interface impairs gating of the severe combined immunodeficiency-related ORAI1 mutant.N端/膜界面处疏水性增加会损害与重症联合免疫缺陷相关的ORAI1突变体的门控功能。
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