Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction
Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction
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DOI：
10.1111/j.1356-9597.2004.00752.x
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发表时间：
2004-07-01
期刊：
影响因子：
2.1
通讯作者：
Nakanishi, S
中科院分区：
文献类型：
--
作者：
Kimura, K;Kitano, J;Nakanishi, S
超极化激活的阳离子电流i-h沿树枝不均匀分布，电流密度随离体体距离的增加而增大。I-h电流的不均匀分布有助于在突触输入的时间整合中归一化位置依赖的变异性，但hcn分布的分子基础仍有待研究。超极化激活的环核苷酸门控阳离子通道位于I-h电流之下，由哺乳动物基因家族的四个成员(HCN1-HCN4)组成。在这项研究中，我们使用了几种不同的方法，包括大鼠脑免疫沉淀、异源表达细胞提取物和谷胱甘肽S转移酶下拉实验，报道了HCN_2与Tamalin、S-SCAM和Mint2支架蛋白形成的蛋白质组装。Tamalin的PDZ结构域与HCN_2在PDZ结合基序和内部羧基末端都有相互作用，而S-SCAM的PDZ结构域的结合发生在HCN_2羧基末端的环核苷酸结合区和CNBD下游序列。Hcn2和Mint2之间的蛋白质组装是由Mint2的Munc18相互作用结构域与Hcn2的CNBD-下游序列相互作用形成的。结果表明，HCN2以不同的蛋白质-蛋白质相互作用模式与多个神经元支架蛋白形成蛋白质复合体。
Hyperpolarization-activated cation currents, termed I-h, are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The non-uniform distribution of I-h currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs) underlie I-h currents and consist of four members (HCN1-HCN4) of the gene family in mammals. In this investigation, we report that HCN2 forms a protein assembly with tamalin, S-SCAM and Mint2 scaffold proteins, using several different approaches including immunoprecipitation of rat brain and heterologously expressing cell extracts and glutathione S-transferase pull-down assays. The PDZ domain of tamalin interacts with HCN2 at both the PDZ-binding motif and the internal carboxy-terminal tail of HCN2, whereas binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD-downstream sequence of the carboxy-terminal tail of HCN2. A protein assembly between HCN2 and Mint2 is formed by the interaction of the munc18-interacting domain of Mint2 with the CNBD-downstream sequence of HCN2. The results demonstrate that HCN2 forms a protein complex with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.