Regulation of ENaC trafficking in rat kidney.
Regulation of ENaC trafficking in rat kidney.
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DOI：
10.1085/jgp.201511533
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发表时间：
2016-03
期刊：
影响因子：
--
通讯作者：
Palmer LG
中科院分区：
文献类型：
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作者：
Frindt G;Gravotta D;Palmer LG
在盐耗竭的反应中，醛固酮驱动Na通道蛋白从ER通过高尔基体移动到肾上皮细胞的顶膜。上皮Na通道（ENaC）形成远端肾单位中Na+重吸收的途径，并且这些通道的调节对于盐稳态是必需的。在大鼠肾脏中，ENaC亚基以未成熟和完全加工的形式到达质膜，后者由内切糖苷酶H不敏感的糖基化或蛋白水解裂解定义。适应低盐饮食的动物具有增加的ENaC表面表达，其对亚基蛋白的成熟形式具有特异性，并且与α、β和γ ENaC相似（三至四倍）。采用差速离心法、蔗糖梯度分离法和免疫吸附法分离肾膜。使用钙连接蛋白抗体分离的内质网膜表达未成熟的γ ENaC，并且含量随着Na耗尽而降低。用抗顺式高尔基体蛋白GM130的抗体分离的高尔基体膜表达未成熟和加工的γ ENaC; Na耗竭使该组分中加工的γ ENaC的含量增加3.8倍。使用抗Rab11抗体分离的内体区室含有未成熟和加工的γ ENaC;加工亚基的含量随着Na消耗增加2.4倍。最后，我们使用尿外泌体间接评估了晚期内吞隔室中γ ENaC的含量。这些外泌体中的所有γ ENaC都是完全裂解的形式，其含量随着Na耗尽而增加4.5倍。这些结果表明，刺激ENaC表面表达的结果，至少部分地从增加率的形成完全加工的亚基在高尔基体和随后的贩运顶端膜。
In response to salt depletion, aldosterone drives the movement of Na channel protein from ER through the Golgi to the apical membrane in kidney epithelial cells. The epithelial Na channel (ENaC) forms a pathway for Na+ reabsorption in the distal nephron, and regulation of these channels is essential for salt homeostasis. In the rat kidney, ENaC subunits reached the plasma membrane in both immature and fully processed forms, the latter defined by either endoglycosidase H–insensitive glycosylation or proteolytic cleavage. Animals adapted to a low-salt diet have increased ENaC surface expression that is specific for the mature forms of the subunit proteins and is similar (three- to fourfold) for α, β, and γENaC. Kidney membranes were fractionated using differential centrifugation, sucrose-gradient separation, and immunoabsorption. Endoplasmic reticulum membranes, isolated using an antibody against calnexin, expressed immature γENaC, and the content decreased with Na depletion. Golgi membranes, isolated with an antibody against the cis-Golgi protein GM130, expressed both immature and processed γENaC; Na depletion increased the content of processed γENaC in this fraction by 3.8-fold. An endosomal compartment isolated using an antibody against Rab11 contained both immature and processed γENaC; the content of processed subunit increased 2.4-fold with Na depletion. Finally, we assessed the content of γENaC in the late endocytic compartments indirectly using urinary exosomes. All of the γENaC in these exosomes was in the fully cleaved form, and its content increased by 4.5-fold with Na depletion. These results imply that stimulation of ENaC surface expression results at least in part from increased rates of formation of fully processed subunits in the Golgi and subsequent trafficking to the apical membrane.