AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2.
We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the beta-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A (PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the beta-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.
Bhalla
Vivek
V
Division of Nephrology, Department of Medicine, University of California at San Francisco, San Francisco, California 94107, USA.
Oyster
Nicholas M
NM
Fitch
Adam C
AC
Wijngaarden
Marjolein A
MA
Neumann
Dietbert
D
Schlattner
Uwe
U
Pearce
David
D
Hallows
Kenneth R
KR
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D004789:Enzyme Activation; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D017868:Cyclic AMP-Dependent Protein Kinases; D056827:Endosomal Sorting Complexes Required for Transport; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D048049:Extracellular Signal-Regulated MAP Kinases; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus