Calmodulin interaction with hEAG1 visualized by FRET microscopy.
Calmodulin interaction with hEAG1 visualized by FRET microscopy.
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DOI：
10.1371/journal.pone.0010873
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发表时间：
2010-05-27
期刊：
影响因子：
3.7
通讯作者：
Stühmer W
中科院分区：
文献类型：
--
作者：
Gonçalves JT;Stühmer W
Ca 2+介导的离子通道调节提供了细胞内信号传导途径和膜电活性之间的联系。细胞内Ca ~（2+）通过与钙调素（CaM）直接结合抑制电压门控钾通道EAG 1。三个CaM结合位点（BD-C1：674-683，BD-C2：711-721，BD-N：151-165）已在肽筛选中鉴定，并被提出介导结合。然而，这三个位点参与钙调素与天然通道的结合仍不清楚。在这里，我们研究了Ca 2 +/CaM的EAG通道的结合，通过可视化YFP标记的CaM和Cerulean标记的hEAG 1在哺乳动物细胞中的相互作用，通过FRET。我们的细胞方法的结果证实，两个钙调素结合位点主要涉及的高亲和力1-8-14为基础的钙调素结合结构域的N-末端和第二个C-末端结合结构域BD-C2。这些位点的突变完全消除了CaM与hEAG 1的结合。我们证明了BD-N和BD-C2结合结构域足以使CaM结合到天然通道，因此BD-C1不能独立地结合CaM。
Ca2+-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca2+ inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. Here we studied the binding of Ca2+/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently.
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