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...

Epithelial Na؉ absorption is regulated by Nedd4-2, an E3 ubiquitin-protein ligase that reduces expression of the epithelial Na ؉ channel ENaC at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 binds to ENaC via PY motifs located in the C termini of ␣-, ␤-, and ␥ENaC. However, little is known about the mechanism by which Nedd4-2 regulates ENaC surface expression. Here we found that Nedd4-2 catalyzes ubiquitination of ␣-, ␤-, and ␥ENaC; Nedd4-2 overexpression increased ubiquitination, whereas Nedd4-2 silencing decreased ubiquitination. Although Nedd4-2 increased both mono/oligoubiquitinated and multiubiquitinated forms of ENaC, monoubiquitination was sufficient for Nedd4-2 to reduce ENaC surface expression and reduce ENaC current. Ubiquitination was disrupted by Liddle syndrome-associated mutations in ENaC or mutation of the catalytic HECT domain in Nedd4-2. Several findings suggest that the interaction between Nedd4-2 and ENaC is localized to the cell surface. First, Nedd4-2 bound to a population of ENaC at the cell surface. Second, Nedd4-2 catalyzed ubiquitination of cell surface ENaC. Third, Nedd4-2 selectively reduced ENaC expression at the cell surface but did not alter the quantity of immature ENaC in the biosynthetic pathway. Finally, Nedd4-2 induced degradation of the cell surface pool of ENaC. Together, the data suggest a model in which Nedd4-2 binds to and ubiquitinates ENaC at the cell surface, which targets surface ENaC for degradation, and thus, reduces epithelial Na ؉ transport.

The dynamics of SARS-CoV-2 replication and shedding in humans remain poorly understood. We captured the dynamics of infectious virus and viral RNA shedding during acute infection through daily longitudinal sampling of 60 individuals for up to 14 days. By fitting mechanistic models, we directly estimated viral expansion and clearance rates, and overall infectiousness for each individual. Significant person-to-person variation in infectious virus shedding suggests that individual-level heterogeneity in viral dynamics contributes to superspreading. Viral genome loads often peaked days earlier in saliva than in nasal swabs, indicating strong tissue compartmentalization and suggesting that saliva may serve as a superior sampling site for early detection of infection. Viral loads and clearance kinetics of Alpha (B.1.1.7) and previously circulating non-variant of concern viruses were mostly indistinguishable, indicating that the enhanced transmissibility of this variant cannot be simply explained by higher viral loads or delayed clearance. These results provide a high-resolution portrait of SARS-CoV-2 infection dynamics and implicate individual-level heterogeneity in infectiousness in superspreading.

As a pathway for Na؉ reabsorption, the epithelial Na ؉ channel ENaC is critical for Na ؉ homeostasis and blood pressure control. Na ؉ transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in ␣, ␤, and ␥ENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved ␣ENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.The epithelial Na ϩ channel ENaC forms a pathway for Na ϩ reabsorption across epithelia, including the kidney, lung, and colon. Therefore, it plays a critical role in Na ϩ homeostasis and blood pressure control (reviewed in Refs. 1 and 2). Defects in ENaC function or regulation cause inherited forms of hypertension and hypotension (3) and may contribute to the pathogenesis of lung disease in cystic fibrosis (4).ENaC is regulated by Nedd4-2, a HECT domain E3 ubiquitin ligase that decreases ENaC expression at the cell surface (5-7). This regulation requires the binding of Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C terminus of each of the three subunits that form the channel (␣, ␤, and ␥ENaC). Mutations in the PY motifs of ␤ or ␥ENaC disrupt binding, causing Liddle syndrome (8 -10). In this inherited form of hypertension, increased expression of ENaC at the cell surface results in excessive renal Na ϩ reabsorption (7,8,11). Binding is also modulated by aldosterone and vasopressin via serum and glucocorticoid-regulated kinase and protein kinase A, respectively; both kinases phosphorylate Nedd4-2, which reduces its binding to ENaC (6,12,13). However, the mechanism by which Nedd4-2 reduces ENaC surface expression is uncertain. It is possible that Nedd4-2 regulates ENaC trafficking in the biosynthetic pathway, targeting it for degradation in the proteasome. Consistent with this model, localization of Nedd4-2 at the cell surface is not required for Nedd4-2 to inhibit ENaC (14). Moreover, proteasome inhibitors decrease ENaC degradation (15-17) and increase ENaC surface expression.2 Alternatively, Nedd4-2 could regulate ENaC in the endocytic pathway, altering ENaC endocytosis and/or...

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