Peter M. Snyder

Inhibition of the Epithelial Na+ Channel by Interaction of Nedd4 with a PY Motif Deleted in Liddle’s Syndrome

The epithelial Na+ channel (ENaC) plays a critical role in Na+ absorption in the kidney and other epithelia. Mutations in the C terminus of the β or γENaC subunits increase renal Na+ absorption, causing Liddle’s syndrome, an inherited form of hypertension. These mutations delete or disrupt a PY motif that was recently shown to interact with Nedd4, a ubiquitin-protein ligase expressed in epithelia. We found that Nedd4 inhibited ENaC when they were coexpressed in Xenopusoocytes. Liddle’s syndrome-associated mutations that prevent the interaction between Nedd4 and ENaC abolished inhibition, suggesting that a direct interaction is required for inhibition by Nedd4. Inhibition also required activity of a ubiquitin ligase domain within the C terminus of Nedd4. Nedd4 had no detectable effect on the single channel properties of ENaC. Rather, Nedd4 decreased cell surface expression of both ENaC and a chimeric protein containing the C terminus of the β subunit. Decreased surface expression resulted from an increase in the rate of degradation of the channel complex. Thus, interaction of Nedd4 with the C terminus of ENaC inhibits Na+ absorption, and loss of this interaction may play a role in the pathogenesis of Liddle’s syndrome and other forms of hypertension.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

A Pore Segment in DEG/ENaC Na+ Channels

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.

Nedd4-2 Induces Endocytosis and Degradation of Proteolytically Cleaved Epithelial Na+ Channels

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 α, β, and γENaC 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 αENaC. 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.

Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits.

The epithelial Na(+) channel (ENaC) regulates Na(+) absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na(+)-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na(+)-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na(+)-channel activity. Previously we showed that a proline-rich region of the alpha subunit of the Na(+) channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na(+)-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the alpha, beta and gamma Na(+)-channel subunits but not to a mutated beta subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na(+)-channel activity.

Down-regulating destruction: phosphorylation regulates the E3 ubiquitin ligase Nedd4-2.

Phosphorylation of Nedd4-2 regulates epithelial Na+ transport. E3 ubiquitin ligases catalyze ubiquitination, which can target specific proteins for degradation. Although a growing number of E3 ubiquitin ligases and their targets have been identified, much less is known about the mechanisms that regulate their activity. A convergence of data indicate that phosphorylation regulates the binding of Nedd4-2, a HECT (homologous to the E6-AP C terminus) domain E3 ubiquitin ligase, to its target, the epithelial Na+ channel ENaC. Nedd4-2 phosphorylation is emerging as a central convergence point for the regulation of epithelial Na+ transport.

Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways

Epithelial Na+ absorption is regulated by Nedd4-2, an E3 ubiquitin 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 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.

A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse α, β, and γ epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of αENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, γENaC contained such a domain just distal to TM2 spanning Gln573–Pro600. Interactive domains were also localized within Met1–Gln54 and the last 17 residues of α- and βENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of αENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of γENaC was established with whole-cell patch clamp experiments of wild type (α, β, and γ) and mutant (α, β, and γΔQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 ± 0.14 versus 2.5 ± 0.80 nA of amiloridesensitive inward current at –80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 ± 0.03 versus 0.67 ± 0.07 for wild type. Mutant γENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573–Pro600, deletion of Gln573–Arg583 but not Thr584–Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573–Arg583 of γENaC are necessary for normal channel gating.

An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2 terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5 variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2 terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na+ transport compared with Sgk1. Together, the data demonstrate that a conserved NH2-terminal variant of Sgk1 shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na+ transport in a heterologous expression system.

Chapter 2 Membrane Topology, Subunit Composition, and Stoichiometry of the Epithelial Na+Channel

Publisher Summary The chapter presents a review of the membrane topology, subunit composition, and stoichiometry of epithelial Na + channel (ENaC). To begin to define the structure of ENaC, the chapter mentions the membrane topology of the rat a subunit (αrENaC). In vitro translation of αrENaC produced a 73-kDa protein, and an additional 93-kDa protein was produced when αrENaC was translated in the presence of microsomal membranes, corresponding to the core-glycosylated form of the protein. Three ENaC subunits have been identified in epithelia of rat and human: α, β, and γ ENaC. It is found that expression of the human, a subunit alone in Xenopus oocytes, produced amiloride-sensitive Na + current. The finding that coexpression of α , β , and γ ENaC is required for maximal Na + current suggests that ENaC is a heteromultimeric channel. Genetic evidence from C. elegans suggests that the degenerins also function as heteromultimers. A biochemical assay is used to determine whether the mass of hENaC was consistent with a complex containing nine subunits. Because α hENaC can form a functional channel by itself, the mass of in vitro is determined, expressed α by sucrose gradient sedimentation.