Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.
Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.
复制标题
阿米洛利敏感的钠通道与肾上皮细胞的细胞骨架相连。
DOI：
10.1073/pnas.88.16.6971
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发表时间：
1991
影响因子：
11.1
通讯作者：
Benos,DJ
中科院分区：
文献类型：
--
作者：
Smith,PR;Saccomani,G;Joe,EH;Angelides,KJ;Benos,DJ
在钠转运的肾上皮细胞中，阿米洛利敏感的钠通道定位于顶膜的微绒毛区。为了阐明维持钠通道在顶膜上分布的因素，我们寻找了与该通道相关的特定蛋白质。Triton X-100提取的A6上皮细胞表明，钠通道与洗涤剂不溶和组装的细胞骨架有关。间接免疫荧光和共聚焦显微镜显示钠通道与顶端微绒毛膜分离，并与锚蛋白、丝蛋白和肌动蛋白共存。我们通过免疫印迹分析证明，从牛肾乳头中分离和纯化后，Ankyrin和Fodrin仍然与钠通道有关。只有在纯化的牛钠通道复合体存在下，抗钠通道抗体才能沉淀~(125)I标记的安吉林。125I标记的锚蛋白的直接结合表明锚蛋白与通道的150 kDa亚基结合。荧光漂白剂的横向扩散测量表明，钠通道的横向迁移率受到严重限制。我们得出结论，Ankyrin将阿米洛利敏感的钠通道连接到细胞骨架，这种联系可能隔离顶端微绒毛的钠通道，并维持它们在肾上皮细胞中的极化分布。
Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergent-insoluble and assembled cytoskeleton. Indirect immunofluorescence and confocal microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 125I-labeled ankyrine can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of 125I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amiloride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells.