Journal
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
Volume 318, Issue 5, Pages F1113-F1121Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00609.2019
Keywords
epithelial Na+ channel; Liddle syndrome; mice; proteolysis; rats
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Funding
- National Institute of Diabetes and Digestive and Kidney Diseases [R01-DK-111380]
- National Institutes of Health and Deutsche Forschungsgemeinschaft (German Research Foundation) [387509280, SFB 1350]
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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-alpha ENaC 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 (similar to 30 kDa) of the subunit. No significant Ub-beta ENaC was detected, indicating that ubiquitination of this subunit is minimal. For gamma ENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (similar to 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 (beta 566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-gamma ENaC 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-alpha ENaC and ub-gamma ENaC 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 aldosteronedependent ENaC upregulation.
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