Surface expression of epithelial Na channel protein in rat kidney.
Surface expression of epithelial Na channel protein in rat kidney.
复制标题
DOI：
10.1085/jgp.200809989
复制
发表时间：
2008-06
影响因子：
3.8
通讯作者：
Palmer, Lawrence G.
中科院分区：
文献类型：
--
作者：
Frindt, Gustavo;Ergonul, Zuhal;Palmer, Lawrence G.
采用肾细胞胞外表面生物素化和膜分离技术研究大鼠肾小管顶膜上皮钠通道（ENaC）蛋白的表达。大鼠肾脏原位灌注含有nhs生物素的溶液，这是一种细胞外生物素衍生物，共价地附着在赖氨酸上的游离氨基上。将膜溶解，用中性微球分离标记蛋白，免疫印迹法检测表面β和γENaC亚基。膜分馏法测定表面αENaC。表面的大部分γENaC分子质量小于全长亚基，这与该亚基在靠近第一跨膜结构域的胞外部分的裂解一致。通过全细胞膜片钳记录，在皮质集管的主要细胞中测量了通道对胰蛋白酶的不敏感性，证实了这一发现。在各种生理条件下，表面均可检测到ENaC亚基。然而，在表面标记前一周，通过低钠饮食或通过渗透微型泵直接注入激素，增加动物体内醛固酮水平，使表面亚基的表达增加了2至5倍。缺钠动物补充盐5 h可降低表面表达。ENaC亚基表面密度的变化显著影响矿化皮质激素对肾细胞内钠转运的调节，但不能完全解释通道活性的增加。
Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.
登录
查看更多内容
DOI：
10.1152/ajprenal.00422.2005
发表时间：
2006-09-01
影响因子：
4.2
作者：
Ergonul, Zuhal;Frindt, Gustavo;Palmer, Lawrence G.
通讯作者：
Palmer, Lawrence G.
DOI：
10.1085/jgp.81.6.785
发表时间：
1983-06
期刊：
The Journal of general physiology
影响因子：
--
作者：
Garty H;Edelman IS
通讯作者：
Edelman IS
DOI：
10.1152/ajplung.00435.2004
发表时间：
2005-05-01
影响因子：
4.9
作者：
Caldwell, RA;Boucher, RC;Stutts, MJ
通讯作者：
Stutts, MJ
影响因子：
5.5
作者：
Caldwell, RA;Boucher, RC;Stutts, MJ
通讯作者：
Stutts, MJ
DOI：
10.1085/jgp.20028559
发表时间：
2002-05
期刊：
The Journal of general physiology
影响因子：
--
作者：
Alvarez de la Rosa D;Li H;Canessa CM
通讯作者：
Canessa CM