Association of the epithelial sodium channel with Apx and α-spectrin in A6 renal epithelial cells
Association of the epithelial sodium channel with Apx and α-spectrin in A6 renal epithelial cells
复制标题
DOI：
10.1074/jbc.274.33.23286
复制
发表时间：
1999-08-13
影响因子：
4.8
通讯作者：
Smith, PR
中科院分区：
文献类型：
--
作者：
Zuckerman, JB;Chen, XY;Smith, PR
最近对上皮钠通道（ENaC）的分子克隆为鉴定调节其细胞表面表达和活性的ENaC相关蛋白提供了机会。我们研究了ENaC是否与爪蟾（Xenopus）肾上皮细胞Apr（顶端蛋白）和基于谱蛋白的膜细胞骨架相关。我们还研究了克隆的ENaC是否需要Apr来表达对酰胺敏感的Na+电流，A6细胞洗涤液提取物的糖密度梯度离心显示xENaC、α -spectrin和Apr共同沉淀。免疫印迹分析在高严格条件下从非洲爪鼠ENaC/ apx梯度组分中提取的蛋白质共免疫沉淀表明，ENaC、Apr和α -spectrin在大分子复合物中是相关的。为了检验ENaC的功能表达是否需要Apr，我们将α - β - γ mENaC crna与Apr正核苷酸或反义寡脱氧核苷酸共注射到非洲爪蟾卵母细胞中。双电极电压钳技术显示，共注射反义寡核苷酸的卵母细胞与共注射义寡核苷酸的卵母细胞相比，阿米洛利敏感电流明显降低。这些研究表明，在A6细胞中，ENaC与Apr和α -谱蛋白以大分子复合物的形式相关，并提示Apr是爪蟾上皮中ENaC功能表达所必需的。
Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apr (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apr is required for the expression of amiloride-sensitive Na+ currents by cloned ENaC, Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apr. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apr, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apr is required for the functional expression of ENaC, alpha beta gamma mENaC cRNAs were coinjected into Xenopus oocytes with Apr sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apr and alpha-spectrin in A6 cells and suggest that Apr is required for the functional expression of ENaC in Xenopus epithelia.