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1 Medicine (Nephrology), Yale University School of Medicine, New Haven, CT, USA
2 Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: john.hayslett{at}yale.edu.
The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells using specific polyclonal antibodies after labeling cells with 35S or 32P. When immune complexes were resolved on SDS-PAGE the 
subunit migrated at 85 and 65 kD, the 
subunit at 115 and 100 kD and the 
subunit at 90 kD. In the resting state all three subunits were phosphorylated. The subunit was phosphorylated only in the 65 kD band, suggesting that the post-translational modification that gives rise to the rapidly migrating form of is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 minutes increased phosphorylation of , and subunits approximately 2-fold. Exposure to 1 µM aldosterone for 16 hrs increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pre-treated with aldosterone, phosphorylation was also increased approximately 2-fold, but the total amount of phosphorylated substrate was larger than in control conditions due to the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, the findings of this study suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport.
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