N-linked glycosylation determines cell surface expression of two-pore-domain K+ channel TRESK
N-linked glycosylation determines cell surface expression of two-pore-domain K+ channel TRESK
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DOI：
10.1016/j.bbrc.2009.12.056
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发表时间：
2010-01-08
影响因子：
3.1
通讯作者：
Doering, Frank
中科院分区：
文献类型：
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作者：
Egenberger, Brigitte;Polleichtner, Georg;Doering, Frank
在小鼠和人TRESK亚基的第一外环内，分别鉴定出一个或两个N-糖基化共有位点。使用定点诱变和Western免疫印迹，发现两种直向同源物的单个残基在异源表达后被糖基化。从爪蟾卵母细胞的双电极电压钳记录显示，N-糖基化突变体的电流幅度降低了80%，相比野生型TRESK。为了研究膜靶向，GFP标记的TRESK亚基在非洲爪蟾卵母细胞中表达，并通过共聚焦显微镜测量细胞表面的荧光强度。N-糖基化突变体的信号降低> 50%，表明它们较低的电流幅度基本上是由于通道的表面表达不足。(C)2009 Elsevier Inc. All rights reserved.
Within the first external loop of mouse and human TRESK subunits one or two N-glycosylation consensus sites were identified, respectively. Using site directed mutagenesis and Western immunoblotting a single residue of both orthologues was found to be glycosylated upon heterologous expression. Two-electrode voltage-clamp recordings from Xenopus oocytes revealed that current amplitudes of N-glycosylation mutants were reduced by 80% as compared to wildtype TRESK. To investigate membrane targeting, GFP-tagged TRESK subunits were expressed in Xenopus oocytes and fluorescence intensity at the cell surface was measured by confocal microscopy. Signals of the N-glycosylation mutants were reduced by >50%, indicating that their lower Current amplitudes substantially result from inadequate surface expression of the channel. (C) 2009 Elsevier Inc. All rights reserved.