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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

¹Department of Medicine (Nephrology) and ²Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut

Submitted 15 January 2004 ; accepted in final form 30 August 2004

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 by 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 kDa, the -subunit at 115 and 100 kDa, and the -subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The -subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of -, -, and -subunits approximately twofold. Exposure to 1 µM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of 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, our findings 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.

epithelial sodium channel; aldosterone; sodium transport

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