The epithelial Na ؉ channel (ENaC) forms the pathway for Na ؉ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na ؉ homeostasis and blood pressure control. Na ؉ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na ؉ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to ␣ENaC, and hence, the hNedd4-2-mediated inhibition of Na ؉ absorption. These data suggest that SGK regulates epithelial Na ؉ absorption in part by modulating the function of hNedd4-2.

The epithelial Na ؉ channel (ENaC) functions as a pathway for epithelial Na ؉ transport, contributing to Na ؉ homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na ؉ transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na ؉ transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na ؉ transport.The transport of Na ϩ across epithelia in the distal nephron, colon, and airway is critical to maintain Na ϩ homeostasis and to control the composition of airway surface liquid. Defects in Na ϩ transport are responsible for genetic and acquired forms of hypertension and hypotension (1) and contribute to the pathogenesis of cystic fibrosis (2-4). Na ϩ transport is mediated by the epithelial Na ϩ channel (ENaC), 1 which forms a pathway for Na ϩ to cross the apical membrane (5, 6). At the basolateral membrane Na ϩ exits the cell via the Na ϩ -K ϩ -ATPase, resulting in transepithelial Na ϩ absorption. Nedd4-2, an E3 ubiquitin-protein ligase, is critical in the control of Na ϩ transport. Nedd4-2 inhibits Na ϩ transport by catalyzing ENaC ubiquitination and degradation, resulting in reduced ENaC expression at the cell surface (for review, see Refs. 7-9). This requires binding of multiple Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C termini of each ENaC subunit (␣, ␤, and ␥). Disruption of this interaction by mutations that delete or alter the PY motifs increases ENaC surface expression and causes Liddle's syndrome, an inherited form of hypertension (10 -14).Recent work suggests that Nedd4-2 is negatively regulated by serum and glucocorticoid-inducible kinase (SGK), a Ser/Thr kinase. SGK binds to Nedd4-2 and phosphorylates it at three sites (see Fig. 3C), which reduces Nedd4-2 binding to ENaC (15, 16). Similar to Liddle's syndrome, decre...

DEG/ENaC Na؉ channels have diverse functions, including Na ؉ absorption, neurotransmission, and sensory transduction. The ability of these channels to discriminate between different ions is critical for their normal function. Several findings suggest that DEG/ ENaC channels have a pore structure similar to K ؉ channels. To test this hypothesis, we examined the accessibility of native and introduced cysteines in the putative P loop of ENaC. We identified residues that span a barrier that excludes amiloride as well as anionic and large methanethiosulfonate reagents from the pore. This segment contains a structural element ((S/G)CS) involved in selectivity of ENaC. The results are not consistent with predictions from the K ؉ channel pore, suggesting that DEG/ENaC Na ؉ channels have a novel pore structure.

Epithelial Na؉ transport is regulated in large part by mechanisms that control expression of the epithelial Na ؉ channel (ENaC) at the cell surface. Nedd4 and Nedd4-2 are candidates to control ENaC surface expression, but it is not known which of these proteins contributes to ENaC regulation in epithelia. To address this question, we used RNA interference to selectively reduce expression of Nedd4 or Nedd4-2. We found that endogenous Nedd4-2, but not Nedd4, negatively regulates ENaC in two epithelial cell lines (Fischer rat thyroid and H441); small interfering RNA (siRNA) against Nedd4-2 increased amiloride-sensitive Na ؉ current (compared with control siRNA), but Nedd4 siRNA did not. A mutation associated with Liddle's syndrome (␤ R566X ) abolished the effect of Nedd4-2 siRNA, suggesting that a defect in ENaC regulation by Nedd4-2 contributes to the pathogenesis of this inherited form of hypertension. Previous work found that Nedd4-2 is phosphorylated by serum and glucocorticoid-regulated kinase, a Ser/Thr kinase induced by steroid hormones. Here we found that Nedd4-2 phosphorylation contributes to ENaC regulation by steroid hormones. Consistent with this model, ENaC stimulation by dexamethasone was reduced by Nedd4-2 siRNA and by overexpression of a mutant Nedd4-2 lacking serum and glucocorticoid-regulated kinase phosphorylation sites. Thus, endogenous Nedd4-2 negatively regulates ENaC in epithelia and is a component of a signaling pathway by which steroid hormones regulate ENaC. Defects in this regulation may contribute to the pathogenesis of hypertension.The rate of Na ϩ transport across a variety of epithelia (e.g. kidney-collecting duct, airway, colon) is regulated in large part by mechanisms that control expression of the epithelial Na1 at the apical cell surface. ENaC, a heteromultimeric complex of three homologous subunits (␣-, ␤-, and ␥-ENaC), forms a pathway for Na ϩ to enter the cell at the apical membrane followed by Na ϩ exit at the basolateral membrane mediated by the Na ϩ -K ϩ -ATPase (1, 2). Defects in ENaC regulation cause blood pressure abnormalities (hypertension or hypotension) (3) and contribute to the pathogenesis of cystic fibrosis (4).Although regulation of ENaC surface expression is critical for Na ϩ homeostasis, little is known about the mechanisms responsible. Previous work (reviewed in Ref. 5) identified Nedd4 and Nedd4-2, HECT WW domain ubiquitin-protein ligases, as candidates. First, Nedd4 and Nedd4-2 are capable of binding to ENaC; multiple WW domains bind to PY motifs located in the C terminus of each ENaC subunit (6 -10). Importantly, mutation of these PY motifs causes an inherited form of hypertension (Liddle's syndrome) (11,12). Second, Nedd4 and Nedd4-2 inhibit ENaC when coexpressed in heterologous cells; a ubiquitin ligase domain catalyzes ENaC ubiquitination, targeting the channel for degradation (9,13,14). As a result, reduced ENaC surface expression decreases Na ϩ current. However, an important limitation is that previous studies tested the effect of Nedd4 and Nedd4-2 over...

Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na ؉ channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na ؉ transport in Liddle's syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high openstate probability, whereas uncleaved channels are inactive. Here, we found that Liddle's syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na ؉ transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na ؉ transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle's syndrome mutations alter ENaC activity.amiloride ͉ hypertension ͉ protease ͉ sodium