LOCALIZATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN CHLORIDE SECRETORY EPITHELIA
LOCALIZATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN CHLORIDE SECRETORY EPITHELIA
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DOI：
10.1172/jci115582
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发表时间：
1992-01-01
影响因子：
15.9
通讯作者：
WELSH, MJ
中科院分区：
文献类型：
--
作者：
DENNING, GM;OSTEDGAARD, LS;WELSH, MJ
囊性纤维化是由囊性纤维化跨膜传导调节因子(CFTR)基因编码突变引起的。为了进一步了解cftr的功能和调控，我们使用共聚焦免疫荧光显微镜对cftr蛋白的不同区域(R(调节)结构域(M13-1)、COOH末端(M1-4)和预测的细胞外结构域(M6-4))进行了定位。所有三种抗体都从表达CFTR的细胞中免疫沉淀出一个155-170-kD的多肽。每种抗体均染色HeLa和3T3细胞，表达重组CFTR，但不表达内源性CFTR的细胞：HeLa、NIH-3T3和内皮细胞。在定位研究中，我们使用了表达内源性CFTR并具有cAMP激活的顶端Cl-通透性的上皮细胞系：T84、CaCo2和HT29克隆19A。我们的结果表明CFTR在这些上皮细胞中是一种顶膜蛋白，因为(A)CFTR的染色类似于几种顶膜标记的染色，但不同于基底膜蛋白的染色；(B)细胞单层的薄片显示在顶膜上染色；以及(C)细胞外域抗体M6-4染色在非通透性细胞的顶面。我们的结果不排除CFTR也位于根尖膜下的可能性。增加细胞内cAMP水平并不改变CFTR的根尖膜染色模式。此外，cAMP介导的根尖膜氯电导增加不需要通过囊泡与根尖膜融合插入通道。这些结果表明CFTR位于分泌氯离子的上皮细胞的顶端质膜上，这一结果与CFTR是顶膜氯离子通道的结论一致。
Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extra-cellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl--secreting epithelia, a result consistent with the conclusion that CFTR is an apical membrane chloride channel.