SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Abstract

Throughout the body, the epithelial Na(+) channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na(+) ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1's inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC's regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.

Figure 1

SPLUNC1, but not other PLUNC family members, inhibits ENaC activity. Current is displayed relative to amiloride-sensitive current from α, β, γENaC-expressing oocytes (ctrl, white bar; n = 18). Oocytes co-expressing SPLUNC1 showed a ∼70% reduction in ENaC current (p < 0.0001; n = 22). However, those co-expressing SPLUNC2 (n = 22) or oocytes co-expressing LPLUN C1 had no significant ENaC current reduction (n = 17). * denotes p < 0.05 different to control oocytes expressing α, β, γ ENaC.

Figure 2

SPLUNC1 is not a protease inhibitor. The trypsin-specific fluorogenic trypsin substrate Boc-Gln-Ala-Arg-MCA, emits fluorescence upon cleavage. Trypsin (1 U/ml, ■); trypsin and aprotinin (1.79 nM, ●); trypsin and SPLUNC1 (1.79 nM, ▲). Boc-Gln-Ala-Arg-MCA alone (○). All n = 4.

Figure 3

SPLUNC1 decreases the number of αENaC subunits in the plasma membrane. (A) Surface biotinylation of αENaC shows that plasma membrane ENaC is decreased following coexpression with SPLUNC1 in Xenopus oocytes. Samples were analyzed by western blot using an anti-V5 (for V5/6His-tagged SPLUNC1 and for V5-CT-tagged αENaC) monoclonal antibody Lane 1, control; Lane 2, αβγENaC; Lane 3, αβγENaC & SPLUNC1. Total lysate per lane was equivalent to 3–4 oocytes and was run on a 10% Gel. (B) Whole cell western blot of oocytes probing for αENaC (top), SPLUNC1 and actin (bottom) shows that total ENaC levels are not decreased by SPLUNC1. Total lysate per lane was equivalent to 3–4 oocytes and was run on a 12% Gel. (C) Addition of MTSET to ENaC containing the βS518C mutant increases ENaC P_o to 1.0 when coexpressed in oocytes yet the overall current is still reduced by SPLUNC1 expression. Open bars, control. Closed bars, MTSET addition, n = 6 for each group. * denotes p < 0.05 different ± MTSET; † denotes p < 0.05 different ± SPLUNC1.