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

2000 ◽  
Vol 345 (3) ◽  
pp. 503-509 ◽  
Author(s):  
Tracy J. FARR ◽  
Sarah J. CODDINGTON-LAWSON ◽  
Peter M. SNYDER ◽  
Fiona J. MCDONALD
Keyword(s):  
Binding Sites ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Channel Activity ◽  
Α Subunit ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Human Lung Cells ◽  
Epithelial Na Channel ◽  
Proline Rich Region

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 α 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 α, β and γ Na+-channel subunits but not to a mutated β subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na+-channel activity.

A single WW domain is the predominant mediator of the interaction between the human ubiquitin-protein ligase Nedd4 and the human epithelial sodium channel

2002 ◽  
Vol 361 (3) ◽  
pp. 481-488 ◽  
Author(s):  
J. Shaun LOTT ◽  
Sarah J. CODDINGTON-LAWSON ◽  
Paul H. TEESDALE-SPITTLE ◽  
Fiona J. MCDONALD
Keyword(s):  
The Body ◽  
Single Amino Acid ◽  
Na Channel ◽  
Α Subunit ◽  
Ww Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Single Amino Acid Change ◽  
Domain 3 ◽  
Neuronal Precursor Cell

The activity of the epithelial Na+ channel (ENaC) is required for the maintenance of salt and water balance in the body. Channel activity is regulated by the ubiquitin-protein ligase Nedd4 ['neuronal precursor cell-expressed developmentally down-regulated (gene 4)'] that interacts with the channel via its WW domains. Mutations in channel subunits that disrupt this interaction cause Liddle's syndrome, a severe inherited form of hypertension. In previous studies we showed that WW domains 2, 3 and 4 of human Nedd4 bound to the human ENaC (hENaC) subunits, whereas WW domain 1 did not. Here we extend this observation to determine the binding affinities of the human Nedd4 WW domains for hENaC C-terminal peptides. We show that WW domains 2, 3 and 4 bind with differing affinities to Na+ channel subunit peptides. WW domain 3 has the highest affinity and we predict that WW domain 3 contributes most of the binding because a construct containing the three WW domains bound no better than WW domain 3 alone. Further, a single amino acid change (Arg165 → Thr) in WW domain 1 enables binding to the α subunit of the channel to occur, with an affinity comparable with that of WW domain 4. Differential binding propensities between the various WW domains and Na+ channel subunit peptides are explained on the basis of quantitative structural modelling of the complexes and their isolated components.

Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na+ channel

2002 ◽  
Vol 283 (3) ◽  
pp. F431-F436 ◽  
Author(s):  
Fiona J. McDonald ◽  
Andrea H. Western ◽  
John D. McNeil ◽  
Brittany C. Thomas ◽  
Diane R. Olson ◽  
...  
Keyword(s):  
Functional Data ◽  
Direct Interaction ◽  
Na Channel ◽  
Critical Component ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Salt And Water Balance ◽  
Protein Ligases ◽  
Epithelial Na Channel ◽  
Genetic Form

The epithelial Na+ channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4–2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na+ transport in epithelia.

Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na+ channel regulation

2001 ◽  
Vol 281 (3) ◽  
pp. F469-F477 ◽  
Author(s):  
Elena Kamynina ◽  
Caroline Tauxe ◽  
Olivier Staub
Keyword(s):  
Close Relative ◽  
Na Channel ◽  
Domain Interaction ◽  
Hect Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Activity Structure ◽  
Py Motif ◽  
Epithelial Na Channel ◽  
Salt Homeostasis

The epithelial Na+ channel (ENaC) is regulated via PY motif-WW domain interaction by the mouse (m) ubiquitin-protein ligase mNedd4-2 but not by its close relative mNedd4-1. Whereas mNedd4-1 is composed of one C2, three WW, and one HECT domain, mNedd4-2 comprises four WW domains and one HECT domain. Both proteins have human (h) homologs, hNedd4-1 and hNedd4-2; however, both of them include four WW domains. Therefore, we characterized hNedd4-1 and hNedd4-2 in Xenopus laevisoocytes with respect to ENaC binding and interaction. We found that hNedd4-2 binds to and abrogates ENaC activity, whereas hNedd4-1 does not coimmunoprecipitate with ENaC and has only modest effects on ENaC activity. Structure-function studies revealed that the C2 domain of hNedd4-1 prevents this protein from downregulating ENaC and that WW domains 3 and 4, involved in interaction with ENaC, do not by themselves provide specificity for ENaC recognition. Taken together, our data demonstrate that hNedd4-2 inhibits ENaC, implying that this protein is a modulator of salt homeostasis, whereas hNedd4-1 is not primarily involved in ENaC regulation.

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes

1992 ◽  
Vol 262 (2) ◽  
pp. G244-G248 ◽  
Author(s):  
C. Asher ◽  
D. Singer ◽  
R. Eren ◽  
O. Yeger ◽  
N. Dascal ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
The Other ◽  
Na Channel ◽  
Channel Activity ◽  
High Affinity ◽  
Other Hand ◽  
Exogenous Rna ◽  
Lower Intestine ◽  
Regulation Of Transport

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

scholarly journals Acute effects of aldosterone on the epithelial Na channel in rat kidney

2015 ◽  
Vol 308 (6) ◽  
pp. F572-F578 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Surface Protein ◽  
Early Response ◽  
Surface Expression ◽  
Na Channel ◽  
Channel Activity ◽  
Acute Effects ◽  
Chronic Stimulation ◽  
Epithelial Na Channels ◽  
Epithelial Na Channel

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 μg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current ( INa). INa averaged 100 pA/cell, 20–30% of that reported for the same preparation under conditions of chronic stimulation. INa was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of β-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

scholarly journals On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+Channel Binding Site

2005 ◽  
Vol 280 (28) ◽  
pp. 26206-26215 ◽  
Author(s):  
Ossama B. Kashlan ◽  
Shaohu Sheng ◽  
Thomas R. Kleyman
Keyword(s):  
Binding Site ◽  
Na Channel ◽  
Α Subunit ◽  
Epithelial Na Channel

Regulation of maturation and processing of ENaC subunits in the rat kidney

2006 ◽  
Vol 291 (3) ◽  
pp. F683-F693 ◽  
Author(s):  
Zuhal Ergonul ◽  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
Salt Restriction ◽  
High K ◽  
Electrophysiological Measurements ◽  
Immature Form ◽  
Epithelial Na Channel

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7–9 days, the abundance of two forms of the α-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the β-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the γ-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in α- and γ-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in α- and γ- but not β-subunits when animals were treated with exogenous aldosterone, and in β- and γ- but not α-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.

Identification of a highly conserved sequence at the N-terminus of the epithelial Na + channel α subunit involved in gating

1999 ◽  
Vol 438 (5) ◽  
pp. 709-715 ◽  
Author(s):  
S. Gründer ◽  
N. Fowler Jaeger ◽  
I. Gautschi ◽  
L. Schild ◽  
B.C. Rossier
Keyword(s):  
Na Channel ◽  
Α Subunit ◽  
Conserved Sequence ◽  
N Terminus ◽  
Epithelial Na Channel

Functional polymorphisms in the α-subunit of the human epithelial Na+ channel increase activity

2006 ◽  
Vol 290 (4) ◽  
pp. F821-F827 ◽  
Author(s):  
Qiusheng Tong ◽  
Anil G. Menon ◽  
James D. Stockand
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Severe Form ◽  
Na Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Ovary Cells ◽  
Functional Polymorphisms ◽  
Black Populations

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in α-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of α-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in α-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered Na+ handling by the kidney.

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