Schönherr R, Löber K, Heinemann S H
Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Strabetae 1, D-07747 Jena, Germany.
EMBO J. 2000 Jul 3;19(13):3263-71. doi: 10.1093/emboj/19.13.3263.
Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.
细胞内钙离子（Ca(2+)）可抑制超快速激活延迟整流钾通道（EAG）家族的电压门控钾通道。为了确定其潜在的分子机制，我们在非洲爪蟾卵母细胞中表达了人类版本的hEAG1。当在无细胞的膜片上进行测量时，这些通道失去了对Ca(2+)的敏感性。然而，通过应用重组钙调蛋白（CaM），Ca(2+)敏感性可以恢复。在存在CaM的情况下，在100 nM Ca(2+)时可使hEAG1通道产生半数抑制。使用标记的CaM和hEAG1的谷胱甘肽S-转移酶（GST）融合片段进行的覆盖分析表明，CaM可直接结合至C末端结构域（hEAG1的第673 - 770位氨基酸）。该区域内的点突变揭示了一个新的CaM结合结构域，推测其形成一个两亲性螺旋，两侧对于结合都很重要。与Ca(2+)激活的钾通道不同，CaM与hEAG1的结合是Ca(2+)依赖性的，其表观解离常数（K(D)）为480 nM。野生型和突变型通道的共表达实验表明，每个通道复合物结合一个CaM分子就足以抑制通道。
