Rab4 GTP/GDP modulates amiloride-sensitive sodium channel (ENaC) function in colonic epithelia
Rab4 GTP/GDP modulates amiloride-sensitive sodium channel (ENaC) function in colonic epithelia
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DOI：
10.1016/j.bbrc.2005.12.036
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发表时间：
2006-02-10
影响因子：
3.1
通讯作者：
George, C
中科院分区：
文献类型：
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作者：
Saxena, SK;Singh, M;George, C
钠选择性阿米洛利敏感上皮钠通道(ENaC)介导致密上皮细胞的电生钠重吸收。ENAC在质膜上的表达需要调节转运。亚基蛋白质的加工和大分子组装以一种明确和高度分隔的方式。RAS相关的RAB GTP酶在高度调控的事件序列中监控这些过程。为了研究Rab蛋白在ENaC功能中的作用，以野生型(WT)突变体Rab4Q67L为研究对象，研究Rab蛋白在ENaC功能中的作用。在结肠癌细胞系中，显性负性GDP锁定突变体Rab4S22N过表达，并记录到对阿米洛利敏感的电流。Rab4过表达抑制阿米洛利敏感电流。通过引入Rab4中和抗体和Rab4特异性siRNA可逆转这一作用。GDP锁定的Rab4突变体抑制而GTPase缺陷突变体适度刺激阿米洛利敏感电流。GTP重叠实验证实了Rab4的活性状态，Western blotting证实了其表达。免疫沉淀和下拉实验表明，Rab4和ENaC之间存在蛋白质-蛋白质相互作用，此外，这种功能调节与ENaC在细胞表面和细胞内池中表达的变化相一致。我们认为Rab4是一个关键元件，通过GTP-GDP状态、循环和表达水平等机制调节ENaC功能。我们的观察表明，上皮细胞系统顶膜上的通道表达结合了RabGTPase作为通道功能的基本决定因素，并为ENaC的治疗增加了一个令人兴奋的范例。(C)2005 Elsevier Inc.保留所有权利。
The sodium-selective amiloride-sensitive epithelial sodium channel (ENaC) mediates electrogenic sodium re-absorption in tight epithelia. ENaC expression at the plasma membrane requires regulated transport. processing, and macromolecular assembly of subunit proteins in a defined and highly compartmentalized manner. Ras-related Rab GTPases monitor these processes in a highly regulated sequence of events. In order to evaluate the role of Rab proteins in ENaC function, Rab4 wild-type (WT), the GTPase-deficient mutant Rab4Q67L. and the dominant negative GDP-locked mutant Rab4S22N were over-expressed in the colon cancer cell line, HT-29 and amiloride-sensitive currents were recorded. Rab4 over-expression inhibited amiloride-sensitive currents. The effect was, reversed by introducing Rab4-neutralizing antibody and Rab4 specific SiRNA. The GDP-locked Rab4 mutant inhibited while GTPase-deficient mutant moderately stimulated amiloride-sensitive currents. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. Immunoprecipitation and pull-down assay suggest protein-protein interaction between Rab4 and ENaC, In addition, the functional modulation coincides with concomitant changes in ENaC expression at the Cell surface and in intracellular pool. We propose that Rab4 is a critical element that regulates ENaC function by mechanisms that include GTP-GDP status, recycling, and expression level. Our observations imply that channel expression in apical membranes of epithelial cell system incorporates RabGTPase as an essential determinant of channel function and adds an exciting paradigm to ENaC therapeutics. (c) 2005 Elsevier Inc. All rights reserved.