Grumbach Yael, Bikard Yann, Suaud Laurence, Chanoux Rebecca A, Rubenstein Ronald C
Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and.
Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Am J Physiol Cell Physiol. 2014 Oct 15;307(8):C701-9. doi: 10.1152/ajpcell.00134.2014.
The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of β-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of β-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.
上皮钠离子通道（ENaC）在血压调节和气道表面液体量的调控中起关键作用。ERp29是一种29 kDa的硫氧还蛋白同源内质网（ER）蛋白，它只有一个半胱氨酸，而非通常硫氧还蛋白的CXXC基序。我们团队之前证明，ERp29可促进囊性纤维化跨膜传导调节因子（CFTR）的生物合成。基于CFTR和ENaC转运的相似性，我们推测ERp29也会调节ENaC的生物合成和功能表达。在上皮细胞中，野生型（wt）ERp29的过表达在尤斯灌流小室实验中增加了ENaC的功能表达[氨氯地平敏感短路电流（Isc）]，以及全细胞裂解物中裂解形式的γ-ENaC的丰度。相反，siRNA介导的ERp29缺失或缺乏其单个半胱氨酸的突变型ERp29（C157S ERp29）的过表达降低了ENaC的功能表达。当将胰蛋白酶应用于顶端表面以激活未裂解的ENaC时，过表达wt ERp29的细胞对氨氯地平敏感Isc的分数增加较小，而过表达C157S ERp29或ERp29缺失的细胞对胰蛋白酶的反应中氨氯地平敏感Isc的分数增加显著更大。有趣的是，这些观察结果与顶端表面β-ENaC表达的改变无关。相反，ERp29似乎促进了β-ENaC与衣被复合体II内质网出口机制的Sec24D货物识别成分的相互作用。总之，这些数据支持以下假设：ERp29将ENaC导向高尔基体，在其生物合成和转运至顶端膜的过程中，ENaC在高尔基体中发生裂解。
