Howard M, Jilling T, DuVall M, Frizzell R A
Department of Physiology and Biophysics, University of Alabama at Birmingham, USA.
Kidney Int. 1996 Jun;49(6):1642-8. doi: 10.1038/ki.1996.239.
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.
囊性纤维化跨膜传导调节因子（CFTR）是一种磷酸化激活的氯离子通道，负责cAMP诱导的氯离子跨上皮细胞顶端膜的分泌。为了在膜定位研究中优化其检测，我们在羧基末端或第四个外环处用表位序列标记CFTR。在两种不同的生理学检测中测试了六种不同标记的CFTR的功能。含有M2表位的CFTR对cAMP有反应，而表达带有血凝素HA标签的CFTR的细胞对cAMP几乎没有反应或没有反应。使用在第四个外环中标记的CFTR，我们证明用福斯可林激活cAMP会导致HeLa细胞质膜中CFTR增加。福斯可林抑制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刺激引起的质膜氯离子传导增加。
