Endothelin-1 Inhibits the Epithelial Na+ Channel through βPix/14-3-3/Nedd4-2
Endothelin-1 Inhibits the Epithelial Na+ Channel through βPix/14-3-3/Nedd4-2
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DOI：
10.1681/asn.2009080885
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发表时间：
2010-05-01
影响因子：
13.6
通讯作者：
Staruschenko, Alexander
中科院分区：
文献类型：
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作者：
Pavlov, Tengis S.;Chandi, Ahmed;Staruschenko, Alexander
上皮性钠离子通道(ENaCs)介导了大脑皮层集合管(CCD)的钠重吸收，但调节这些通道活性的调控途径还不完全清楚。在这里，我们观察到内皮素-1(ET-1)通过减少通道的开放概率而显著地减弱ENaC的活性，并通过减少质膜中的通道数量来慢性地减弱ENaC的活性。为了研究ET-1激活的信号蛋白β(1)Pix是否介导ENaC活性，我们在有或没有共表达β(1)Pix的CHO细胞中重组了ENaC，发现β(1)Pix对ENaC具有负性调节作用。敲除天然主细胞中的βPix可阻断ET-1引起的ENaC通道数目的减少。此外，我们还发现，βPix并不通过其对rac1和cc42的鸟嘌呤核苷酸交换因子活性来降低ENaC的活性。相反，共表达的β(1)Pix突变结构揭示了Beta(1)Pix通过结合14-3-3蛋白影响ENaC活性。免疫共沉淀实验支持培养的主细胞中β(1)Pix和14-3-3β之间的物理相互作用。共表达14-3-3β可增加CHO细胞的ENaC活性，但同时表达β(1)Pix可抑制这种增加。14-3-3β被β(1)Pix募集，削弱了14-3-3β与泛素连接酶Nedd4-2的相互作用，从而促进了ENaC的泛素化和降解。综上所述，这些结果提示，慢性ET-1对ENaC的抑制作用是由βPix与14-3-3/Nedd4-2途径相互作用所致。
Epithelial Na+ channels (ENaCs) mediate sodium reabsorption in the cortical collecting duct (CCD), but the regulatory pathways that modulate the activity of these channels are incompletely understood. Here, we observed that endothelin-1 (ET-1) attenuates ENaC activity acutely by reducing the channel's open probability and chronically by decreasing the number of channels in the plasma membrane. To investigate whether beta(1)Pix, a signaling protein activated by ET-1, mediates ENaC activity, we reconstituted ENaC in CHO cells with or without coexpressed beta(1)Pix and found that beta(1)Pix negatively regulates ENaC. Knockdown of beta Pix in native principal cells abolished the ET-1-induced decrease in ENaC channel number. Furthermore, we found that beta Pix does not decrease ENaC activity through its guanine nucleotide exchange factor (GEF) activity for Rac1 and Cdc42. Instead, coexpression of beta(1)Pix mutant constructs revealed that beta(1)Pix affects ENaC activity through binding 14-3-3 proteins. Coimmunoprecipitation experiments supported a physical interaction between beta(1)Pix and 14-3-3 beta in cultured principal cells. Coexpression of 14-3-3 beta increased ENaC activity in CHO cells, but concomitant expression of beta(1)Pix attenuated this increase. Recruitment of 14-3-3 beta by beta(1)Pix impaired the interaction of 14-3-3 beta with the ubiquitin ligase Nedd4-2, thereby promoting ubiquitination and degradation of ENaC. Taken together, these results suggest that the inhibitory effects of chronic ET-1 on ENaC result from beta Pix interacting with the 14-3-3/Nedd4-2 pathway.