Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.
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.
Michlig
Stéphanie
S
Département de Pharmacologie et de Toxicologie, Université de Lausanne, 27, rue du Bugnon, CH-1005 Lausanne, Switzerland.
Harris
Michael
M
Loffing
Johannes
J
Rossier
Bernard C
BC
Firsov
Dmitri
D
D000450:Aldosterone / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D015222:Sodium Channels / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000502:physiology*; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D014981:Xenopus; D029867:Xenopus Proteins
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; C493554:SLC34A1 protein, human; D015222:Sodium Channels; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D025801:Ubiquitin; D029867:Xenopus Proteins; D014443:Tyrosine; D000450:Aldosterone; D011374:Progesterone; D000584:Amiloride; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D048049:Extracellular Signal-Regulated MAP Kinases; D010100:Oxygen