Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits.
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ABSTRACT:
SUBMITTER: Farr TJ
PROVIDER: S-EPMC1220784 | biostudies-other | 2000 Feb
REPOSITORIES: biostudies-other
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Project description:Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney's long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2's ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.
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Project description:Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.
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Project description:The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.
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Project description:The epithelial Na+ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na+ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCD(cl4) cells, displaying ENaC-dependent transepithelial Na+ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na+ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.
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Project description:The epithelial Na+ channel (ENaC) possesses a large extracellular domain formed by a β-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, β, or γ). Removing the γ subunit thumb domain had no effect on Na+ currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or β subunit thumb domain exhibited significantly reduced Na+ currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na+ self-inhibition response, whereas this response was retained in channels lacking a β thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature β subunit and mature γ subunit was observed when the β or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na+ Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.
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Project description:Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming ? subunit and one or more ? subunits. There are five mammalian ? subunits in total: ?1, ?1B, ?2, ?3, and ?4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, ?1B, the ? subunits are type I topology transmembrane proteins. In contrast, ?1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC ? subunits are multifunctional. While they do not form the ion channel pore, ? subunits alter gating, voltage-dependence, and kinetics of VGSC? subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, ? subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. ? subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of ? subunits is ?1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and ?-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. ? subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. ? subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some ? subunit functions may operate independently of ? subunits. Thus, ? subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.
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Project description:Epithelial Na(+) channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel beta subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na(+) channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.
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Project description:The E3 ubiquitin ligase Nedd4-2 regulates several ion transport proteins, including the epithelial Na(+) channel (ENaC). Nedd4-2 decreases apical membrane expression and activity of ENaC. Although it is subject to tight hormonal control, the mechanistic basis of Nedd4-2 regulation remains poorly understood. To characterize regulatory inputs to Nedd4-2 function, we screened for novel sites of Nedd4-2 phosphorylation using tandem mass spectrometry. Three of seven identified Xenopus Nedd4-2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for SGK1, whereas four were novel, including Ser-293, which matched the consensus for a MAPK target sequence. Further in vitro and in vivo phosphorylation experiments revealed that Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or ERK1/2. Additional rounds of tandem mass spectrometry identified two other phosphorylated residues within Nedd4-2, including Thr-899, which is present within the catalytic domain. Nedd4-2 with mutations at these sites had markedly inhibited JNK1-dependent phosphorylation, virtually no ENaC inhibitory activity, and significantly reduced ubiquitin ligase activity. These data identify phosphorylatable residues that activate Nedd4-2 and may work together with residues targeted by inhibitory kinases (e.g. SGK1 and protein kinase A) to govern Nedd4-2 regulation of epithelial ion transport.
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Project description:Mutations in genes encoding subunits of the epithelial Na+ channel (ENaC) can cause early onset familial hypertension, demonstrating the importance of this channel in modulating blood pressure. It remains unclear whether other genetic variants resulting in subtler alterations of channel function result in hypertension or altered sensitivity of blood pressure to dietary salt. This study sought to identify functional human ENaC variants to examine how these variants alter channel activity and to explore whether these variants are associated with altered sensitivity of blood pressure to dietary salt. Six-hundred participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study with salt-sensitive or salt-resistant blood pressure underwent sequencing of the genes encoding ENaC subunits. Functional effects of identified variants were examined in a Xenopus oocyte expression system. Variants that increased channel activity included three in the gene encoding the ?-subunit (?S115N, ?R476W, and ?V481M), one in the ?-subunit (?S635N), and one in the ?-subunit (?L438Q). One ?-subunit variant (?A334T) and one ?-subunit variant (?D31N) decreased channel activity. Several ?-subunit extracellular domain variants altered channel inhibition by extracellular Na+ (Na+ self-inhibition). One variant (?A334T) decreased and one (?V481M) increased cell surface expression. Association between these variants and salt sensitivity did not reach statistical significance. This study identifies novel functional human ENaC variants and demonstrates that some variants alter channel cell surface expression and/or Na+ self-inhibition.
| S-EPMC5130461 | biostudies-literature
Project description:Canonical epithelial sodium channels (ENaCs) are heterotrimers formed by ?, ?, and ? ENaC subunits in vertebrates and belong to the Degenerin/ENaC family of proteins. Proteins from this family form mechanosensitive channels throughout the animal kingdom. Activity of canonical ENaC is regulated by shear force (SF) mediating Na+ absorption in the kidney and vascular tone of arteries. Expression analysis suggests that non-canonical ENaC, formed by single or only two subunits, exist in certain tissues, but it is unknown if these channels respond to SF. ?, ?, ?, and ? ENaC subunits were expressed either alone or in combinations of two subunits in Xenopus oocytes. Amiloride-sensitive currents and the responses to SF were assessed using two-electrode voltage clamp recordings. With the exception of ? ENaC, all homomeric channels provided amiloride-sensitive currents and responded to SF applied via a fluid stream directed onto the oocytes. Channels containing two subunits were also activated by SF. Here, the presence of the ? ENaC subunit when co-expressed with ? or ? augmented the SF response in comparison to the ???/??? ENaC. Overall, we provide evidence that non-canonical ENaC can form channels that respond to SF. This supports a potential function of non-canonical ENaC as mechanosensors in epithelial, vascular, and sensory cells.
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