Shi Shujie, Buck Teresa M, Nickerson Andrew J, Brodsky Jeffrey L, Kleyman Thomas R
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
Deparment of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania.
Am J Physiol Cell Physiol. 2022 Jan 1;322(1):C111-C121. doi: 10.1152/ajpcell.00335.2021. Epub 2021 Dec 1.
The mammalian paraoxonases (PONs) have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2, and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in fisher rat thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts before ENaC subunit ubiquitination. As a result of enhanced ENaC subunit ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirects the channel for ER-associated degradation.
哺乳动物对氧磷酶（PONs）与抵御氧化应激有关。然而，该家族成员（PON1、PON2和PON3）的生理作用仍在研究之中。PON2和PON3在肾脏的醛固酮敏感远端肾单位中表达，并已证明对上皮钠通道（ENaC）的表达具有负调控作用，ENaC是一种三聚体离子通道，负责协调盐和水平衡。迄今为止，尚未探究这种现象的本质。因此，为了研究PON2调节ENaC的机制，我们在Fisher大鼠甲状腺（FRT）细胞中同时表达PON2和ENaC亚基，该系统适合对ENaC组装和运输进行生化分析。我们发现，PON2主要存在于FRT细胞的内质网（ER）中，其表达降低了每个ENaC亚基的丰度，这反映出亚基周转加快。相比之下，对编码ENaC亚基的mRNA水平没有明显影响。用氯喹或NH4Cl抑制溶酶体功能并没有改变PON2对ENaC表达的抑制作用。相反，PON2以蛋白酶体依赖的方式加速ENaC降解，并在ENaC亚基泛素化之前发挥作用。由于ENaC亚基泛素化和降解增强，PON2降低了FRT细胞中通道的表面表达以及ENaC介导的Na+转运。总之，我们的数据表明，PON2作为一种内质网伴侣蛋白，监测ENaC的生物合成，并将该通道重定向至内质网相关降解途径。
