Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits
Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits
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DOI：
10.1152/ajpheart.00052.2004
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发表时间：
2004-08-01
影响因子：
4.8
通讯作者：
Zhou, ZF
中科院分区：
文献类型：
--
作者：
Gong, QM;Anderson, CL;Zhou, ZF
人类以太-a-go-go相关基因（HERG）的突变导致长QT综合征。我们之前的研究表明，表达为同四聚体通道的HERG N470D突变会导致蛋白质运输缺陷，这可以通过HERG通道阻断药物E-4031来纠正。据报道，当N470D突变体与野生型通道亚基共表达时，也会导致HERG电流的显性负抑制。本研究的目的是：1)探讨N470D突变体与野生型亚基共表达的显性负作用的分子机制；2)检验E-4031是否可以从药理学上挽救运输缺陷的异质通道。结合免疫沉淀和Western blot方法，我们发现N470D突变体和野生型HERG亚基在内质网中以异质通道的形式存在物理关联。这种聚集导致了内质网中野生型和N470D亚基的保留。在E-4031中培养细胞增加了这些通道的细胞表面表达，尽管电生理表型发生了改变。这些结果表明，N470D野生型共组装通道的主要负面作用是由内质网中异质通道的保留引起的，这些通道的运输缺陷可以通过特定的药理策略来纠正。
Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies.