A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels
A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels
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DOI：
10.1073/pnas.0900180106
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发表时间：
2009-08-04
影响因子：
11.1
通讯作者：
Trudeau, Matthew C.
中科院分区：
文献类型：
--
作者：
Gustina, Ahleah S.;Trudeau, Matthew C.
人类ethera-go-go相关基因（hERG）钾通道在心脏复极化中发挥核心作用，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，这是一种灾难性的心律失常。N-末端结构域和通道的成孔“核心”之间的相互作用被提出来调节失活，然而，尽管其至关重要，但失活的机制基础尚不清楚。在这里，为了更直接地研究失活的调节机制，我们将N-末端结构域与荧光蛋白基因融合，并用电生理学和蛋白质相互作用与福斯特共振能量转移（FRET）光谱测试通道功能。hERG N-末端区域的截短显著加速失活，在此我们报告重新应用编码N-末端残基1-135（“eag结构域”）的基因片段足以恢复失活的调节。我们发现，荧光团标记的eag结构域和N-截短的通道是在质膜上的FRET确定的紧密接近。具有Y 43 A或R56 Q（LQTS基因座）突变的eag结构域表现出较少的失活调节和较少的FRET，而eag结构域恢复对全长Y 43 A或R56 Q通道的失活门控的调节并表现出FRET。这项研究表明，直接的，非共价相互作用之间的eag域和通道的核心是足以调节失活门控，LQTS突变扰动的eag域和通道之间的物理相互作用，和小分子，如eag域代表一种新的方法恢复功能的通道致病突变。
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 electrophysiology 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.