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Control of ENaC ubiquitination.

Shujie Shi1, Gustavo Frindt2, Sarah Christine M Whelan1

  • 1Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States.

American Journal of Physiology. Renal Physiology
|June 13, 2024
PubMed
Summary

The ubiquitination of the epithelial sodium channel (ENaC) γ subunit is determined by its cellular location, not its cleavage state. Increased intracellular sodium levels can also stimulate ENaC ubiquitination, impacting channel expression.

Keywords:
FRT cellsfeedback inhibitionkidneyproteolytic cleavage

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Area of Science:

  • Cellular Biology
  • Molecular Physiology
  • Membrane Protein Regulation

Background:

  • Ubiquitination is a key post-translational modification regulating protein stability and trafficking.
  • The epithelial sodium channel (ENaC) plays a critical role in sodium homeostasis.
  • Selective ubiquitination of the mature, cleaved form of the ENaC γ subunit has been previously reported.

Purpose of the Study:

  • To elucidate the mechanisms governing the selective ubiquitination of the ENaC γ subunit.
  • To determine whether protein cleavage or cellular location dictates γENaC ubiquitination.
  • To investigate the impact of intracellular sodium levels on ENaC ubiquitination.

Main Methods:

  • Utilized native rodent kidneys and heterologously expressing Fisher rat thyroid (FRT) cells.
  • Studied wild-type and cleavage-site abolished γENaC mutants coexpressed with α- and βENaC.
  • Employed in situ surface biotinylation and clathrin-mediated endocytosis inhibition (Dyngo-4a).
  • Assessed ubiquitination levels under varying intracellular sodium conditions (amiloride, monensin, Na+ repletion).

Main Results:

  • Both singly and fully cleaved γENaCs were strongly ubiquitinated, indicating cleavage is not essential.
  • Ubiquitination occurred when cleavage-site abolished mutants were coexpressed with other ENaC subunits, facilitating Golgi trafficking.
  • Ubiquitination occurred in both apical and subapical fractions, suggesting location near the apical membrane is important.
  • Inhibition of endocytosis increased cell surface and ubiquitinated γENaC.
  • Increased intracellular sodium (via monensin or Na+ repletion) enhanced γENaC ubiquitination and decreased ENaC expression.

Conclusions:

  • γENaC ubiquitination specificity is primarily determined by its cellular location, particularly in or near the apical membrane, rather than its cleavage state.
  • Increased intracellular sodium concentration can stimulate γENaC ubiquitination, leading to reduced overall ENaC expression.
  • These findings provide insights into the regulation of ENaC activity and expression through ubiquitination-dependent pathways.