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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels
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A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels

机译：重组的N末端结构域可完全恢复N截短和长QT综合征突变体hERG钾通道的失活门控

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摘要

Human ether a go-go related gene (hERG) potassium channels play a central role in cardiac repolarization where channel closing (deactivation) regulates current density during action potentials. Consequently, mutations in hERG that perturb deactivation are linked to long QT syndrome (LQTS), a catastrophic cardiac arrhythmia. Interactions between an N-terminal domain and the pore-forming "core" of the channel were proposed to regulate deactivation, however, despite its central importance the mechanistic basis for deactivation is unclear. Here, to more directly examine the mechanism for regulation of deactivation, we genetically fused N-terminal domains to fluorescent proteins and tested channel function with electrophysi-ology and protein interactions with Forster resonance energy transfer (FRET) spectroscopy. Truncation of hERG N-terminal regions markedly sped deactivation, and here we report that reapplication of gene fragments encoding N-terminal residues 1-135 (the "eag domain") was sufficient to restore regulation of deactivation. We show that fluorophore-tagged eag domains and N-truncated channels were in close proximity at the plasma membrane as determined with FRET. The eag domains with Y43A or R56Q (a LQTS locus) mutations showed less regulation of deactivation and less FRET, whereas eag domains restored regulation of deactivation gating to full-length Y43A or R56Q channels and showed FRET. This study demonstrates that direct, noncovalent interactions between the eag domain and the channel core were sufficient to regulate deactivation gating, that an LQTS mutation perturbed physical interactions between the eag domain and the channel, and that small molecules such as the eag domain represent a novel method for restoring function to channels with disease-causing mutations.
机译：人体活动相关基因（hERG）钾通道在心脏复极中起着核心作用，其中通道关闭（失活）调节动作电位期间的电流密度。因此，干扰失活的hERG突变与长QT综合征（LQTS）有关，后者是灾难性的心律不齐。 N末端域和通道的成孔“核心”之间的相互作用被提出来调节失活，但是，尽管它具有至关重要的作用，但失活的机理基础尚不清楚。在这里，为了更直接地检查失活调节的机制，我们将N末端域遗传融合到荧光蛋白上，并通过电生理学和蛋白相互作用（通过Forster共振能量转移（FRET）光谱）测试了通道功能。 hERG N末端区域的截断显着加速了失活，在这里我们报道了编码N末端残基1-135（“ Eag域”）的基因片段的重新应用足以恢复失活的调节。我们显示荧光标记的eag域和N截短的通道在质膜上非常接近，如FRET所确定。具有Y43A或R56Q（一个LQTS基因座）突变的eag域显示较少的失活调节和较少的FRET，而eag域恢复了失活的调节门控至全长Y43A或R56Q通道并显示FRET。这项研究表明，eag结构域和通道核心之间的直接，非共价相互作用足以调节失活门控，LQTS突变扰动了eag结构域和通道之间的物理相互作用，而小分子（例如eag结构域）代表了一种新型恢复具有致病突变的通道的功能的方法。

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  • 作者

    Ahleah S. Gustina; Matthew C. Trudeau;

  • 作者单位

    Program in Neuroscience, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201 Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201;

    Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    eag domain; FRET; LQTS;

    机译：eag域;烦恼;轻量级;

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