Investigation of RING domain E3 ubiquitin ligases in regulation of delta epithelial sodium channel (δENaC) activity

Abstract

The delta epithelial sodium channel subunit (δENaC), is a member of ENaC family of channels that are essential for sodium homeostasis and blood pressure regulation. Although the role of δENaC has yet to be established it is postulated to be involved in Na+ reabsorption and nociception, and is likely to be subjected to regulation at the gene and protein levels.

Down-regulation of surface proteins via the ubiquitination pathway is a well-known phenomenon, selection of proteins to be modified with ubiquitin for degradation require the association of an E3 ubiquitin ligase. The proline-tyrosine (PY)-motif found in the α, β, and γENaC subunits is required for interaction with HECT E3 ligases. By contrast, δENaC does not contain a conserved PY motif, therefore in this study regulation of δENaC by RING E3 ubiquitin ligases was investigated.

A positive regulator, X-linked inhibitor of apoptosis (XIAP), and a negative regulator, neuronal precursor cell expressed developmentally downregulated gene 8 (Nedd8), of δβγENaC were identified. Further studies showed XIAP and Nedd8 also regulated the αβγENaC channel.

Nedd8 (a ubiquitin-like protein) is known to modulate the function of multi subunit RING domain E3 ligases such as the SCF ubiquitin ligase. Nedd8 functions as a negative regulator of ENaC channels containing αβγ- or δβγENaC by enhancing ubiquitination of δENaC, ENaC and γENaC, and thereby reducing the cell surface population of δβγENaC and γENaC. Consequently, a reduction in functional activity of δβγENaC or γENaC was observed as a decrease in amiloride-sensitive Na+ current in mammalian epithelia expressing δβγENaC or γENaC in the presence of Nedd8. This pathway of action was confirmed by inhibiting Nedd8 conjugation with MLN4924, and this resulted in decreased δENaC ubiquitination and an increased δENaC cell surface population.

In contrast, the single subunit RING E3 XIAP was shown to decrease ubiquitin modification of δENaC, although this was dose-dependent and only apparent with low amounts of XIAP. Reduction in ubiquitination of δENaC by XIAP led to enhanced overall expression of δENaC proteins. As expected, an increase in amiloride-sensitive Na+ current was observed in mammalian epithelia co-expressing δβγENaC and XIAP. XIAP was confirmed to downregulate COMMD1, a negative regulator of δENaC, therefore XIAP’s effect may be indirect through promoting degradation of COMMD1. This was confirmed by functional experiments measuring δβγENaC Na+ transport in the presence of COMMD1 siRNA, and in the absence or presence of XIAP. When COMMD1 was knocked down, XIAP could no longer increase the amiloride-sensitive Na+ current.

Nedd8 or COMMD1 regulation of δENaC could involve promoting the activity of one or more Nedd8-dependent-RING E3 ligase complexes to down-regulate δENaC, as COMMD1 has been shown to enhance the activity of Nedd8-RING E3 ligase complexes. MALDI mass spectrometry analysis of Nedd8-δENaC coimmunoprecipitates was performed to identify other components of the putative RING-E3 ligase, such as F-box proteins, that might bring δENaC to a RING E3 complex. However no F-box proteins were identified. 14-3-3 proteins were identified in the proteomic screen and coimmunoprecipitation analysis showed δENaC interacts with 14-3-3 proteins. As an alternative mechanism, ENaC may interact with 14-3-3 proteins and these could promote binding of an F-box protein to δENaC bringing δENaC to an SCF complex for ubiquitination.

This study identified two RING E3 ligases that regulate δENaC via the ubiquitination pathway, with Nedd8 having predominately negative effects on δENaC, and XIAP showing positive effects on δENaC. This project furthers our understanding of the regulation of δ- and α- containing ENaC via RING E3 ubiquitination regulatory pathways.