DISTINCT EFFECTS OF GLUTATHIONE DISULFIDE ON δ‐ AND α‐EPITHELIAL SODIUM CHANNEL SUBUNITS

Abstract

BackgroundAmiloride‐sensitive epithelial sodium channel (ENaC) is a membrane bound ion channel with selective inward permeability to Na. ENaC plays a critically important role in maintaining net fluid homeostasis. To date four ENaC subunits (α‐, β‐, γ‐, and δ‐ENaC) have been cloned in mammalian species. Mice, however, only express αβγ. In humans, an additional ENaC subunit (δ1) and its slice variant (δ2) have been recently identified. Unlike αβγ‐ENaC, the physiological role and regulation of channels made up of δ1 and δ2 subunits remain unclear.HypothesisWe tested the hypothesis that sodium channels containing α and δ ENaC subunits (alongside β and γ) are differentially regulated under oxidative stress.MethodsUsing the Xenopus laevis oocyte expression system, full‐length cDNAs for human ENaC subunits, and two electrode voltage clamp measurements, we modeled antioxidant depletion (a form of oxidative stress) and compared the effects of oxidized glutathione (GSSG) on αβγ‐ENaC and δβγ‐ENaC whole cell current. High extracellular GSSG treatments (400μM) increases the redox potential, and models antioxidant depletion in an oocyte expression system. This experimental approach allows for the singular study of αβγ vs. δβγ ENaC in a cell model system capable of normal assembly of multimeric ion channels into the membrane.ResultsGSSG significantly decreased ENaC activity in oocytes expressing αβγ‐ENaC (n=10). Conversely, GSSG significantly increased whole cell current in oocytes expressing δ1βγ‐ENaC (n=12) and in δ2βγ‐ENaC expressing oocytes (n=13) immediately upon extracellular exposure. While both α and δ ENaC subunits are directly modified by GSSG at Cys thiol sites, the effect and mechanism of GSSG posttranslational modification of α vs δ expressing Na channels differed significantly in our studies. Heterologous expression of αβR566Xγ ENaC in oocytes indicate that GSSG proteolytically degrades classical αβγ ENaC. Whole cell recordings and biotinylation studies indicate that δ2 ENaC subunits increase net Na transport via insertion of new membrane protein, whereas GSSG plays no role in δ1 transport and insertion into the cell membrane.ConclusionWe found that αβγ ENaC activity is decreased by GSSG. Conversely δβγ ENaC activity is significantly increased by GSSG. This conclusion highlights the need to better understand the opposing translational effects of α vs δ ENaC subunits, as it may occur in human tissue. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of α and δ ENaC subunits.Support or Funding InformationSupport for this project was made possible by the University of Utah Investigator Initiated Grant awarded to MNH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.