Article: article from journal or magazin.
Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.
Journal of Biological Chemistry
Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.
Aldosterone/pharmacology, Amiloride/pharmacology, Amino Acid Motifs, Animals, Binding Sites, Biotinylation, Catalysis, Cell Membrane/metabolism, Down-Regulation, Endosomal Sorting Complexes Required for Transport, Epithelial Sodium Channel, Extracellular Signal-Regulated MAP Kinases/metabolism, Humans, MAP Kinase Signaling System, Mutation, Oocytes/metabolism, Oxygen/metabolism, Patch-Clamp Techniques, Phosphorylation, Progesterone/chemistry, Progesterone/metabolism, Protein Binding, Protein Structure, Tertiary, RNA, Complementary/metabolism, Rats, Signal Transduction, Sodium Channels/metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIa/physiology, Time Factors, Tyrosine/chemistry, Ubiquitin/chemistry, Ubiquitin-Protein Ligases/physiology, Xenopus
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