Department of Clinical Research, University of Bern, Switzerland.
J Mol Cell Cardiol. 2011 Jul;51(1):90-8. doi: 10.1016/j.yjmcc.2011.03.015. Epub 2011 Apr 2.
The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.
电压门控心肌钾通道 hERG1（人类 ether-à-go-go 相关基因 1）在心脏动作电位（AP）的复极化阶段发挥关键作用。其基因 KCNH2 的突变可导致通道生物合成和成熟缺陷，导致先天性长 QT 综合征（LQTS）。为了确定调节 hERG1 通道在质膜上密度的分子机制，我们研究了泛素连接酶 Nedd4-2 对 hERG1 通道的泛素化，这是一种先前与其他离子通道相关的翻译后调节机制。我们发现，在 HEK293 细胞中记录的全细胞 hERG1 电流在神经前体细胞表达发育下调 4-2（Nedd4-2）共表达时会降低。通过 Western blot 和生物素化测定分别评估总 HEK293 裂解物和细胞表面的 hERG1 通道量，发现它们同时减少。Nedd4-2 和 hERG1 通过位于 hERG1 羧基末端的 PY 基序相互作用。最后，我们确定 Nedd4-2 介导 hERG1 的泛素化，并且该基序的缺失会影响 Nedd4-2 依赖性调节。这些结果表明，Nedd4-2 对 hERG1 蛋白的泛素化及其随后的下调可能是调节人类心脏动作电位持续时间的重要机制。
