Knight Kristin K, Wentzlaff Danielle M, Snyder Peter M
Departments of Internal Medicine and Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
Departments of Internal Medicine and Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
J Biol Chem. 2008 Oct 10;283(41):27477-27482. doi: 10.1074/jbc.M804176200. Epub 2008 Jul 28.
Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).
上皮细胞对钠离子（Na⁺）的转运部分是由上皮钠离子通道（ENaC）介导的。先前的研究表明，Na⁺是ENaC的重要调节因子，可提供一种负反馈机制来维持Na⁺的稳态。ENaC以无活性前体的形式合成，通过α和γ亚基细胞外结构域的蛋白水解切割而被激活。在此，我们发现Na⁺部分通过改变该通道的蛋白水解激活来调节ENaC。当Na⁺浓度较低时，我们发现细胞表面的大多数ENaC处于切割/活性状态。随着Na⁺浓度增加，ENaC的切割呈剂量依赖性降低，因此ENaC活性也降低。这种Na⁺效应依赖于Na⁺的通透；ENaC阻滞剂氨氯吡咪以及降低ENaC活性的突变（α（H69A））会增加切割，而激活ENaC的突变（β（S520K））则会减少切割。此外，Na⁺离子载体莫能菌素可逆转失活突变（α（H69A））对ENaC切割的影响，这表明细胞内Na⁺调节切割。Na⁺不会改变Nedd4-2（一种调节ENaC切割的E3泛素连接酶）的活性，但Na⁺可减少外源性胰蛋白酶对ENaC的切割。我们的研究结果支持这样一种模型，即细胞内Na⁺通过改变ENaC切割位点对蛋白酶的可及性来调节切割，并为早期观察到的细胞内Na⁺通过ENaC抑制Na⁺转运（Na⁺反馈抑制）提供了分子解释。
