AMP-activated Kinase Inhibits the Epithelial Na+ Channel through Functional Regulation of the Ubiquitin Ligase Nedd4-2

Abstract

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 ␤-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 ␤-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.The epithelial Na ϩ channel (ENaC) 2 is an apical Na ϩ channel expressed in a variety of salt-reabsorbing epithelial tissues, including the kidney, lung, exocrine gland ducts, and colon (1). This channel plays a major role in the regulation of total body salt and volume homeostasis, blood pressure, and airway surface liquid clearance. ENaC is comprised of three structurally similar ␣-, ␤-, and ␥-subunits, whose full-length forms are ϳ90 -95 kDa in size. Each subunit has two presumed transmembrane domains, a large extracellular loop, and cytoplasmic N and C termini (1). ENaC expressed in oocytes has a presumed ␣ 2 ␤ 1 ␥ 1 stoichiometry (2, 3), although alternate subunit stoichiometries have been proposed (4, 5). ENaC can be identified by its sensitivity to the diuretic amiloride, its ohmic currentvoltage relationship, and its high selectivity for conductance of Na ϩ and Li ϩ over K ϩ (1). ENaC is regulated by the actions of several hormones, including aldosterone, vasopressin, and insulin, as well as various non-hormonal mechanisms. Cellular mechanisms that control ENaC activity include the regulation of channel synthesis, intracellular trafficking, membrane insertion and retrieval, and single channel properties (1). In addition, rece...

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.…”

“…As the E3 ubiquitinprotein ligase Nedd4-2 has emerged as a central locus for the regulation of ENaC expression at the apical plasma membrane (21), we considered that ENaC regulation by IKK␤ may be mediated indirectly through Nedd4-2. Of note, Nedd4-2 has been recently reported to be a substrate for various kinases, including SGK1, PKA, AMPK, and Grk2, which may modulate Nedd4-2 function (3,4,20,22). To test whether IKK␤ phosphorylates Nedd4-2 in vitro, FLAG-xNedd4-2 was expressed in and immunoprecipitated from HEK-293 cell lysates, and in vitro phosphorylation assays were performed (Fig.…”

“…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. 7.…”

“…Assays of Nedd4-2 in vitro phosphorylation were performed as described previously (20). HEK-293T cells transiently transfected with 3 g of plasmid DNA/60-mm dish using Lipofectamine 2000 (Invitrogen) to express wild-type or mutant FLAG-xNedd4-2 were lysed 1 day after transfection.…”

“…In Vivo Phosphorylation Assays-In vivo phosphorylation assays were performed based on our previously described protocol (20). Either Mam-X (stably transfected with a short hair-pin RNA that does not target any known mammalian gene), HEK-293 cells, or HEK-293T cells were transiently co-transfected using Lipofectamine 2000 with the pMO-FLAGxNedd4-2 plasmid and either SMARTpool IKK␤ siRNA or siGENOME non-targeting siRNA according to the manufacturer's instructions 2 days prior to experimentation.…”