Oxidized Glutathione Increases Delta-Subunit Expressing Epithelial Sodium Channel Activity in Xenopus laevis Oocytes
Oxidized Glutathione Increases Delta-Subunit Expressing Epithelial Sodium Channel Activity in Xenopus laevis Oocytes
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发表时间：
2020-05
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通讯作者：
Garett J. Grant;Camila Coca;Xing-Ming Zhao;My N. Helms
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作者：
Garett J. Grant;Camila Coca;Xing-Ming Zhao;My N. Helms
上皮钠通道（ENaC）是由α、β和γ亚基组成的异源三聚体结构，在维持体液平衡中起重要作用。当δ-ENaC亚基代替α-ENaC亚基与β-和γ-亚基一起表达时，可以观察到ENaC生物物理性质的根本变化。利用人ENaC cRNA构建体和Xenopas laevis卵母细胞表达系统，我们发现氧化型谷胱甘肽（GSSG）对αβγ-ENaC和αβγ-ENaC电流的影响不同。GSSG（400 μM）显著降低表达αβγ-ENaC的卵母细胞的标准化全细胞电流，相反增加表达δ1βγ-ENaC和δ2βγ-ENaC的卵母细胞的全细胞电流。GSSG处理增加了表达所有四种亚基的卵母细胞中的电流。Western blot和PCR分析表明，人小气道上皮细胞（hSAEC）表达αβγ亚基和δ ENaC亚基。hSAEC对GSSG单通道反应的差异表明气道上皮细胞氧化还原敏感性可能取决于δ或α亚基是否组装在膜中。计算机模拟分析预测δ-ENaC胞外环中的6个Cys氨基酸和N-末端结构域中的单个Cys对GSSG的翻译后修饰敏感。需要更多的研究来更好地了解氧化型谷胱甘肽和δ-ENaC在肺部疾病中的分子调节和病理生理作用。
Epithelial sodium channels (ENaC) are heterotrimeric structures, made up of α, β, and γ subunits, and play an important role in maintaining fluid homeostasis. When δ-ENaC subunits are expressed in place of (or in addition to) the α-ENaC subunit alongside β- and γ- subunits, fundamental changes in the biophysical properties of ENaC can be observed. Using human ENaC cRNA constructs and the Xenopas laevis oocyte expression system, we show that oxidized glutathione (GSSG) differently effects αβγ-ENaC and αβγ-ENaC current. GSSG (400 μM) significantly decreased normalized whole cell current in oocytes expressing αβγ-ENaC, and conversely increased whole cell current in δ1βγ-ENaC and δ2βγ-ENaC expressing oocytes. GSSG treatment increased current in oocytes expressing all four subunits. Western blot and PCR analysis show that human small airway epithelial cells (hSAEC) express canonical αβγ-subunits alongside δ-ENaC subunits. Differences in single channel responses to GSSG in hSAECs indicate that airway epithelia redox sensitivity may depend on whether δ- or α- subunits assemble in the membrane. In silico analysis predict that six Cys amino acids in the δ-ENaC extracellular loop, and a single Cys in the N-terminal domain, are susceptible to post-translational modification by GSSG. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of oxidized glutathione and δ-ENaC in lung disorders.