Ubiquitination of renal ENaC subunits in vivo
Frindt G, Bertog M, Korbmacher C, Palmer LG (2020)
Publication Type: Journal article
Publication year: 2020
Journal
Book Volume: 318
Pages Range: F1113-F1121
Journal Issue: 5
DOI: 10.1152/ajprenal.00609.2019
Abstract
Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40-70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-βENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90-150 kDa). In mice with a Liddle syndrome mutation (β566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.
Authors with CRIS profile
Marko Bertog
Lehrstuhl für Physiologie (Vegetative Physiologie)
Christoph Korbmacher
Lehrstuhl für Physiologie (Vegetative Physiologie)
Involved external institutions
United States (USA) (US)How to cite
APA:
Frindt, G., Bertog, M., Korbmacher, C., & Palmer, L.G. (2020). Ubiquitination of renal ENaC subunits in vivo. American Journal of Physiology-Renal Physiology, 318(5), F1113-F1121. https://doi.org/10.1152/ajprenal.00609.2019
MLA:
Frindt, Gustavo, et al. "Ubiquitination of renal ENaC subunits in vivo." American Journal of Physiology-Renal Physiology 318.5 (2020): F1113-F1121.
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