The epithelial sodium channel (ENaC) is a key regulator of salt homeostasis. The classic ENaC consists of three subunits: α, β and γ, which are highly expressed in the kidney and colon where they mediate electrogenic Na⁺ influx into cells under the tight hormonal regulation of aldosterone.
A fourth ENaC subunit named [delta]ENaC also generates Na⁺ influx with the β- and γENaC subunits in Xenopus oocytes. However [delta]ENaC differs to the other subunits in its channel properties and tissue distribution, suggesting that [delta]ENaC may possess a physiological role other than salt regulation. A copper-toxicosis related protein called COMMD1/Murr1 was previously identified to directly interact with [delta]ENaC and downregulate [delta]ENaC activity. COMMD1 is linked with multiple ubiquitination pathways, therefore we hypothesised that COMMD1 directly interacts with [delta]ENaC through novel protein-protein interaction motifs and promotes internalisation of [delta]ENaC from the cell surface through enhanced ubiquitination.
With the use of GST pulldown assays and coimmunoprecipitation, it was found that the binding of COMMD1 to [delta]ENaC is mediated by the COMM domain of COMMD1, primarily through amino acids 120-150 of COMMD1. Immunocytochemical studies showed that the intracellular interaction between [delta]ENaC and COMMD1 predominantly occurred in the early and recycling endosomes, suggesting that COMMD1 may promote the retrieval of [delta]ENaC from the cell surface to the intracellular pool. COMMD1 mediated a decrease in the [delta]ENaC cell surface population, as shown by a biotinylation surface labelling assay. This may be driven by an ubiquitin-regulated endocytosis, as COMMD1 increased ubiquitination, but not proteasomal/lysosomal degradation, of [delta]ENaC. COMMD1 may promote [delta]ENaC ubiquitination through the action of the ubiquitin ligase Nedd4-2 as coexpression with Nedd4-2 enhanced the COMMD1-mediated decrease in surface [delta]ENaC expression. This is abolished by the addition of the Nedd4-2 downregulator kinase sgk1, suggesting that COMMD1 may downregulate [delta]ENaC through the Nedd4-2/sgk1 pathway. Surface levels of [delta]ENaC may also be affected by XIAP, a RING domain ubiquitin ligase which is able to decrease the levels of COMMD1. Coimmunoprecipitation of endogenous [delta]ENaC and COMMD1 proteins, and the enhanced colocalisation of endogenous [delta]ENaC in the recycling endosomes with transfected COMMD1, indicate that interaction between transfected [delta]ENaC and COMMD1 reflect the intracellular interactions of the endogenous proteins.
Taken together, these findings suggest that COMMD1 downregulates [delta]ENaC activity by promoting the internalisation of surface [delta]ENaC into early and recycling endosomes and this may be mediated by enhanced [delta]ENaC ubiquitination via the ubiquitin ligase Nedd4-2.
| Identifer | oai:union.ndltd.org:ADTP/217868 |
| Date | January 2008 |
| Creators | Chang, Chi-Chun, n/a |
| Publisher | University of Otago. Department of Physiology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Chi-Chun Chang |
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