Control of ENaC ubiquitination

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.