Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels
Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels
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DOI：
10.1073/pnas.1201997109
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发表时间：
2012-05-15
影响因子：
11.1
通讯作者：
Zagotta, William N.
中科院分区：
文献类型：
--
作者：
Bankston, John R.;Camp, Stacey S.;Zagotta, William N.
离子通道作为大分子复合物的一部分在完整的组织中起作用，大分子复合物包括细胞骨架蛋白、支架蛋白、信号分子和一系列其他分子。组成这些复合物的蛋白质可以影响通道的运输、定位和生物物理性质。TRIP8b（含rab8b的四肽重复相互作用蛋白）是最近发现的超极化激活环核苷酸门控（HCN）通道的辅助亚基，它有助于许多类型神经元中HCN通道的大量树突定位。TRIP8b与HCN通道的羧基末端区相互作用，调节其细胞表面表达水平和环核苷酸依赖性。本文研究了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性4:4复合物。荧光检测大小排斥色谱法和荧光各向异性使我们确认，在TRIP8b的羧基末端部分，有两个不同的结构域（四磷酸甲苷重复区和TRIP8b保守区）分别与HCN羧基末端的两个不同区域相互作用：羧基末端三氨基酸（SNL）和环核苷酸结合结构域。最后，利用x射线晶体学，我们确定了TRIP8b与HCN2的羧基末端肽络合物的四萜区域的原子结构。总之，这些实验开始揭示TRIP8b结合和HCN通道调控的机制。
Ion channels operate in intact tissues as part of large macromolecular 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 the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.