title: Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells.
abstract: The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Carattino
authors.forename: Robert S
authors.initials: RS
authors.lastname: Edinger
authors.forename: Heather J
authors.initials: HJ
authors.lastname: Grieser
authors.forename: Rosalee
authors.initials: R
authors.lastname: Wise
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: John P
authors.initials: JP
authors.lastname: Johnson
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
mesh_terms: D055372:AMP-Activated Protein Kinases; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D053143:Caspase 2; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D016764:Cell Polarity / Q000187:drug effects / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D053503:Epithelial Sodium Channels; D005260:Female; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009097:Multienzyme Complexes / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology; D014981:Xenopus
chemical_list: D053503:Epithelial Sodium Channels; D009097:Multienzyme Complexes; D015222:Sodium Channels; D000584:Amiloride; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D053143:Caspase 2; D020169:Caspases