Identification of a protein-protein interaction between KCNE1 and the activation gate machinery of KCNQ1.
Identification of a protein-protein interaction between KCNE1 and the activation gate machinery of KCNQ1.
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DOI：
10.1085/jgp.200910386
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发表时间：
2010-06
期刊：
影响因子：
--
通讯作者：
Kobertz WR
中科院分区：
文献类型：
--
作者：
Lvov A;Gage SD;Berrios VM;Kobertz WR
KCNQ 1通道与KCNE 1跨膜（TM）肽组装形成具有缓慢激活门开放的电压门控K+通道复合物。与KCNE 1 TM片段相邻的胞质C-末端结构域与调节KCNQ 1门控有关，但其与KCNQ 1的相互作用尚未被描述。在这里，我们鉴定了KCNE 1 C-末端结构域与KCNQ 1 S6激活门和S4-S5接头之间的蛋白质-蛋白质相互作用。使用半胱氨酸交联，我们在KCNQ 1-KCNE 1复合物中生化筛选了300多个半胱氨酸对，并确定了KCNQ 1中与KCNE 1 C-末端结构域中的半胱氨酸残基形成二硫键的三个残基（H363 C，P369 C和I257 C）。交联效率的统计分析表明，H363 C优先与KCNE 1残基H73 C，S74 C和D 76 C反应，而P369 C仅显示出对D 76 C的偏好。电生理研究的突变体K+通道复合物显示，KCNQ 1残基，H363 C，形成交联不仅与KCNE 1亚基，但也与邻近的KCNQ 1亚基的复合物。涉及H363 C残基的交联形成是状态依赖性的，主要发生在KCNQ 1-KCNE 1复合物关闭时。基于这些生化和电生理数据，我们生成了KCNQ 1-KCNE 1胞质区域的闭合状态模型，其中这些蛋白质-蛋白质相互作用准备减缓激活门的打开。
KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K+ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K+ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.
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