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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels
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Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels

机译：辅助亚基TRIP8b与超极化激活的环状核苷酸门控通道相互作用的结构和化学计量

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摘要

Ion channels operate in intact tissues as part of large macromolec-ular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a single-molecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b-the tetratricopepide repeat region and the TRIP8b conserved region-interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotide-binding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of thecarboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism forTRIP8b binding and regulation of HCN channels.
机译：离子通道作为大分子复合物的一部分在完整的组织中运作，这些大分子复合物可包括细胞骨架蛋白，支架蛋白，信号分子和其他分子的连串。组成这些复合物的蛋白质会影响通道的运输，定位和生物物理特性。 TRIP8b（含四三肽重复序列的Rab8b相互作用蛋白）是最近发现的超极化激活的环状核苷酸门控（HCN）通道的辅助亚基，有助于许多类型的神经元中HCN通道的实质树突定位。 TRIP8b与HCN通道的羧基末端区域相互作用，并调节其细胞表面表达水平和环状核苷酸依赖性。在这里，我们研究了TRIP8b与HCN2通道结合的分子决定因素。使用单分子荧光漂白方法，我们发现TRIP8b和HCN2在完整通道中形成专一的4：4复合物。荧光检测尺寸排阻色谱法和荧光各向异性使我们能够确认，在TRIP8b的羧基末端部分（四三肽重复区和TRIP8b保守区）的两个不同结构域与HCN羧基末端区域的两个不同区域相互作用：羧基末端的三个氨基酸（SNL）和环状核苷酸结合结构域。最后，使用X射线晶体学，我们确定了TRIP8b的四三肽区域与HCN2羧基末端的肽复合的原子结构。总之，这些实验开始揭示TRIP8b结合和调节HCN通道的机制。

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    John R. Bankston; Stacey S. Camp; Frank DiMaio; Alan S. Lewis; Dane M. Chetkovich; William N. Zagotta;

  • 作者单位

    Department of Physiology and Biophysics, and University of Washington School of Medicine, Seattle, WA 98195;

    Department of Physiology and Biophysics, and University of Washington School of Medicine, Seattle, WA 98195;

    Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195;

    The Ken and Ruth Davee Department of Neurology and Clinical Neurosciences, and Northwestern University Feinberg School of Medicine,Chicago, IL 60611;

    The Ken and Ruth Davee Department of Neurology and Clinical Neurosciences, and Northwestern University Feinberg School of Medicine,Chicago, IL 60611, Department of Physiology, Northwestern University Feinberg School of Medicine,Chicago, IL 60611;

    Department of Physiology and Biophysics, and University of Washington School of Medicine, Seattle, WA 98195;

  • 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

    beta subunit; single molecule; protein-protein interaction;

    机译：β亚基单分子蛋白质相互作用;

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