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 voltageclamp 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 ϳ1. 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 AMPKdependentinhibition.ThesefindingsindicatethatAMPKdependent 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.The maintenance of transmembrane ion and solute gradients is a crucial process that permits normal cellular functioning, viability, and coordinated transepithelial transport but also consumes a substantial portion of total cellular energy (1). Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including ENaC 1 are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them (2). However, the cellular mechanisms that link membrane transport to energy production and metabolic status have remained elusive.AMPK is a ubiquitous metabolic-sensing Ser/Thr kinase that exists as a heterotrimer composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. Its activity increases during conditions of metabolic stress, in response to elevated intracellular AMP:ATP ratios (3). A parallel activation pathway involves phosphorylation of the ␣ subunit by a recently identified upstream LKB1 kinase complex at a Thr residue in its activation loop (4). The earliest discovered substrates of AMPK were important ...

Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

“…When monolayers reached a transepithelial resistance Ͼ1000 ohm-cm 2 (22), cells were incubated in serum-free, supplement-free medium for 24 h prior to treatment. Following treatment for 3 h with ethanol (vehicle) or 1 M aldosterone (Sigma), cells were lysed in 150 l of lysis buffer (10 mM Tris-HCl (pH 7.4), 50 mM EDTA, 0.4% deoxycholate acid, and 1% Nonidet P-40) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM, benzamidine, and 1ϫ Complete Protease Inhibitor Mixture (Roche Applied Science)) and phosphatase inhibitors (25 mM sodium fluoride, 2 M microcystin-LR, and 1ϫ Phosphatase-Inhibitor Mixture Sets I and II (Calbiochem)).…”

Functional Regulation of the Epithelial Na+ Channel by IκB Kinase-β Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2

“…However, it is not yet clear what the potential upstream regulators of the IKK pathway are in this case. Interestingly, the metabolic sensor AMPK has been reported to be an upstream inhibitor of IKK in several cell systems (37)(38)(39), and we have shown that AMPK inhibits ENaC, also via effects on Nedd4-2 in an AMPK phosphorylation-dependent manner (7,20). Further studies to test for the potential involvement of AMPK and other cellular signaling pathways as upstream regulators of the IKK␤-dependent regulation of ENaC are thus warranted.…”

“…Plasmids expressing NH 2 -terminal hemagglutinin-tagged and COOH-terminal V5-tagged ENaC subunits were kindly provided by Dr. Thomas Kleyman. HEK-293 and mpkCCD c14 cells were cultured in high glucose, Dulbecco's modified Eagle's medium and defined medium supplemented with hormones and nutrients, respectively, as described previously (7).…”

“…Oocyte Two-electrode Voltage Clamp Measurements-Xenopus laevis oocytes were harvested, collagenase-treated, and maintained as described previously (7). Oocytes were injected with cRNAs encoding mouse ␣-, ␤-, and ␥-ENaC (7), NH 2 -terminal FLAG-xNedd4-2 (20), and/or IKK␤ (10) cRNAs in the combinations and amounts indicated in the legend to Fig.…”

“…Several kinases have been shown to be important in mediating these regulatory influences, including SGK1, protein kinase A (PKA), protein kinase C, ERK, the G-protein-coupled receptor kinase (Grk2), and AMP-activated protein kinase (AMPK) (1,(3)(4)(5)(6)(7)(8). In some cases, as with ERK and Grk2, these kinases phosphorylate ENaC directly (5,6); but in many cases, kinase activity alters the activity of key intracellular regulatory proteins that control the apical membrane insertion or retrieval of ENaC (1,9).…”