Abstract
The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of beta and gamma subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.
Highlights
The cortical collecting tubule of the kidney exhibits a rapid increase in sodium permeability after stimulation with aldosterone [1]
In the cortical collecting tubule of the rat and rabbit [10] and in the A6 cell line derived from the distal tubule of the kidney of Xenopus laevis [11], aldosterone increases the levels of sgk mRNA and protein within 30 min after addition of the hormone
Role of Serine and Threonine Residues in the Carboxyl Terminus of  Subunits in the Response to sgk—We have previously shown that ENaCs are phosphorylated in the carboxyl termini of the  and ␥ subunits under basal conditions and that aldosterone induces additional phosphorylation mainly in the  subunit in transfected Madin-Darby canine kidney cells [12]
Summary
The cortical collecting tubule of the kidney exhibits a rapid increase in sodium permeability after stimulation with aldosterone [1]. In the cortical collecting tubule of the rat and rabbit [10] and in the A6 cell line derived from the distal tubule of the kidney of Xenopus laevis [11], aldosterone increases the levels of sgk mRNA and protein within 30 min after addition of the hormone This rapid induction suggests that sgk may mediate the early increase of sodium permeability characteristically observed in the cortical collecting tubule after stimulation with aldosterone. We used Xenopus oocytes co-injected with cRNAs encoding ENaC and sgk as our experimental model This strategy provides an approach to examine the activity of channels with electrophysiological techniques and to measure expression of channel proteins with biochemical methods. The validity of this model system is based on the observation that sgk increases the magnitude of amiloride-sensitive currents in oocytes [10, 11], indicating that these cells have the machinery necessary to mediate the sgk response
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