cAMP-regulated trafficking of epitope-tagged CFTR
cAMP-regulated trafficking of epitope-tagged CFTR
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DOI：
10.1038/ki.1996.239
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发表时间：
1996-06-01
影响因子：
19.6
通讯作者：
Frizzell, RA
中科院分区：
文献类型：
--
作者：
Howard, M;Jilling, T;Frizzell, RA
囊性纤维化跨膜电导调节器 (CFTR) 是一种磷酸化激活的氯离子通道，负责 cAMP 诱导的 Cl 分泌穿过上皮细胞的顶膜。为了优化其在膜定位研究中的检测，我们在羧基末端或第四个外部环中用表位序列标记 CFTR。在两种不同的生理测定中测试了六种不同标记的 CFTR 的功能。含有 M2 表位的 CFTR 对 cAMP 作出反应，而表达带有血凝素 HA 标签的 CFTR 的细胞几乎没有或没有 cAMP 反应。使用第四个外部环中标记的 CFTR，我们证明使用毛喉素激活 cAMP 会导致 HeLa 细胞质膜中 CFTR 的增加。 Forskolin 抑制 CFTR 内吞作用，这有助于细胞表面 CFTR 表达的增加。我们的结果表明，细胞表面 CFTR 的调节有助于 cAMP 刺激引起的质膜 Cl 电导的增加。
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel responsible for cAMP-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection in membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. The function of six different tagged-CFTRs was tested in two different physiological assays. CFTRs containing the M2 epitope responded to cAMP, whereas cells expressing CFTR with the hemagglutinin HA tag showed little or no cAMP response. Using CFTR tagged in the fourth external loop, we demonstrate that cAMP activation using forskolin results in an increase in CFTR in the plasma membrane of HeLa cells. Forskolin inhibited CFTR endocytosis, and this contributes to the increase in cell surface CFTR expression. Our results indicate that regulation of cell surface CFTR contributes to the increase in plasma membrane Cl conductance evoked by cAMP stimulation.