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J. Biol. Chem., Vol. 280, Issue 18, 17608-17616, May 6, 2005

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Epithelial Sodium Channel Inhibition by AMP-activated Protein Kinase in Oocytes and Polarized Renal Epithelial Cells^*

Marcelo D. Carattino, Robert S. Edinger, Heather J. Grieser, Rosalee Wise, Dietbert Neumann¶, Uwe Schlattner¶, John P. Johnson, Thomas R. Kleyman, and Kenneth R. Hallows||

From the Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the ^¶Institute of Cell Biology, HPM D23, Swiss Federal Institute of Technology, 8093 Zurich, Switzerland

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 -mENaC subunit (S518K) along with wild type and increased the channel open probability (P_o) to 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 -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.

Received for publication, February 16, 2005

^* This work was supported by National Institutes of Health Grants R01 DK047874 and DK057718 (to J. P. J.), R01 DK054354 (to T. R. K.), and K08 DK059477 and R03 DK068390 (to K. R. H.) and the Swiss Society for Research on Muscle Diseases and Swiss National Science Foundation Grant 3100A0-102075 (to U. S. and T. Wallimann). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a postdoctoral fellowship award from the Pennsylvania-Delaware Affiliate of the American Heart Association.

^|| To whom correspondence should be addressed: Renal-Electrolyte Division, Dept. of Medicine, University of Pittsburgh School of Medicine, S976 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9580; Fax: 412-383-8956; E-mail: hallows{at}pitt.edu.

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