Kristensen B, Birkelund S, Jorgensen P L
Biomembrane Center, The August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen OE, Denmark.
J Membr Biol. 2003 Jan 1;191(1):25-36. doi: 10.1007/s00232-002-1043-3.
Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.
将增强型绿色荧光蛋白（EGFP）融合到大鼠Na，K-ATP酶α1亚基的C末端，这是一种用于可视化活COS-1肾细胞中Na，K泵在PKA或PKC刺激下转运的新方法。使用嘌呤霉素和哇巴因联合选择程序，在COS-1细胞中实现了荧光嵌合体（Na，K-EGFP）的稳定功能性表达。与EGFP融合后，无论是在基础状态还是调节状态下，Na，K泵的活性均未改变。在共聚焦激光扫描显微镜和荧光显微镜下，Na，K-EGFP嵌合体主要分布在COS细胞的质膜上。在未受刺激的COS细胞中，Na，K-EGFP也存在于溶酶体和从内质网到质膜途中的囊泡中，但在用荧光转铁蛋白标记的回收内体中几乎不存在。激活蛋白激酶A或C后，共定位的Na，K-EGFP和转铁蛋白囊泡的密度增加了3至4倍，而哇巴因敏感的86Rb摄取减少了22%。同时激活PKA和PKC具有累加效应，共定位增加6倍，86Rb摄取减少38%。在用冈田酸抑制蛋白磷酸酶后，观察到了相似程度的反应。通过回收内体中的内化作用减少表面质膜中Na，K-ATP酶的量，可能部分解释了蛋白激酶激活或蛋白磷酸酶抑制后Na，K泵活性的降低。
