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The role of individual Nedd4–2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels¹

ANDREW B. FOTIA^*,, ANUWAT DINUDOM, KEITH E. SHEARWIN, JAN-PETER KOCH^||, CHRISTOPH KORBMACHER^||,, DAVID I. COOK and SHARAD KUMAR^*,2

^* Hanson Institute, Adelaide, Australia;
Department of Medicine and
Department of Molecular Biosciences, Adelaide University, Adelaide, Australia;
Department of Physiology, University of Sydney, Sydney, NSW 2006, Australia;
^|| University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, UK; and
Institut für Zelluläre und Molekulare Physiologie, Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany

²Correspondence: Hanson Institute, IMVS, PO Box 14, Rundle Mall, Adelaide, SA 5000, Australia. E-mail: sharad.kumar{at}imvs.sa.gov.au

SPECIFIC AIMS

All three subunits of the amiloride-sensitive epithelial sodium channel (ENaC) contain PPxY motifs that interact with the WW domains of the ubiquitin-protein ligases Nedd4 and Nedd4–2. This interaction is necessary to mediate ubiquitination-dependent down-regulation of ENaC activity under high Na⁺. The main aim of the current study was to analyze the requirements of individual WW domains of Nedd4–2 in binding to specific ENaC subunits and in the regulation of ENaC function. We also report the identification of two novel Nedd4–2 isoforms.

PRINCIPAL FINDINGS

1. Two novel tissue-specific isoforms of Nedd4–2
We have identified two differentially spliced Nedd4–2 transcripts in mice and humans. The two transcripts originate due to differential splicing of the 60 bp coding exon 7 of the gene encoding residues 236–255 of the Nedd4–2 protein. We have named the shorter isoform Nedd4–220, as it lacks 20 amino acid residues that lie just before the WW2 of Nedd4–2 protein. RT-PCR analysis of the two isoforms indicated that although some tissues, such as gut and lung, expressed both splice variants, the longer Nedd4–2 isoform was predominantly expressed in gut, lung, and kidney, whereas Nedd4–220 was present in most tissues examined except kidney. To test whether Nedd4–2 and Nedd4–220 differ in their ability to inhibit ENaC function, we used the Xenopus oocyte system. Coexpression of the three ENaC subunits resulted in large amiloride-sensitive whole-cell inward currents with I_ami averaging 16.8 ± 1.0 µA at a holding potential of -60 mV. Coexpression of Nedd4–220 or Nedd4–2 reduced I_ami on average by 90.9 ± 2.2% or 85.6 ± 2.4%, respectively. In contrast, coexpression of the catalytically inactive C/S mutants that lack ubiquitin-protein ligase activity of Nedd4–220 or Nedd4–2 had no significant effect on I_ami. These results suggest that both Nedd4–2 isoforms have the ability to regulate ENaC.

2. Nedd4–2 WW3 and WW4 bind all three ENaC subunits
Nedd4–2 contains 4 WW domains. To test which of the Nedd4–2 WW domains mediate interaction with the ENaC subunits, we generated GST fusion proteins containing individual WW domains and their inactive mutants. Equal amounts of the affinity-purified GST-WW domain fusion proteins were immobilized on nitrocellulose membranes after separation by SDS-PAGE and probed with the ³²P-labeled carboxyl termini of mouse ENaC subunits. A recombinant protein containing all four WW domains of mouse Nedd4–2 interacted with all three ENaC subunits (Fig. 1 ). All three ENaC subunits bound to WW3 and WW4 strongly, whereas no significant binding was seen to WW1 and WW2 (Fig. 1) . None of the WW domain mutant proteins showed any binding, indicating that the signals in far-Western blots represent specific WW domain–ligand interactions (Fig. 1) . To further characterize the relative affinities of interactions between Nedd4–2 WW domains and ENaC subunits we used surface plasmon resonance (SPR) analysis. The interactions exhibited very rapid on and off rates, precluding analysis on the basis of kinetics. Data were instead analyzed on the basis of the steady-state response. Consistent with the far-Western results, binding of WW1 to all of the subunits showed a response no greater than that seen with the same concentration of GST alone, indicating that WW1 binding to the ENaC subunits is extremely weak. WW2 binding to the ENaC subunits gave a response only just greater than that observed for GST. WW3 and WW4 bound all three ENaC subunits relatively strongly, binding by WW3 being consistently stronger than binding by WW4. Dissociation constants for WW3 and WW4 binding were in the high nanomolar to low micromolar range. The order of affinity of WW domains for ENaC subunits can be summarized as WW3 > WW4 > WW2 > WW1. There appeared to be little discrimination between the three ENaC subunits by the WW domains, although WW4 bound slightly better to than to ß or .

View larger version (63K): [in this window] [in a new window]   Figure 1. Far-Western analysis of binding between Nedd4–2 WW domains and ENaC subunits. 2 µg of each affinity purified GST WW fusion protein was used in far-Western assays. A) Coomassie blue-stained WT and mutant (mut) GST-WW proteins. Proteins were transferred to nitrocellulose membranes and hybridized to 32P-labeled ENaC (B), ßENaC (C), or ENaC (D) probes. Molecular mass markers in kDa are indicated.

3. Nedd4–2 WW domains 3 and 4 are required for the down-regulation of ENaC
To investigate which of the WW domains of Nedd4–2 are required to prevent inhibition of ENaC by high intracellular Na⁺, we used the whole-cell patch clamp technique with single mouse mandibular duct cells. A pipette solution rich in Na⁺ (70 mM) leads to almost complete inhibition of the amiloride-sensitive current, which is mediated by Nedd4/Nedd4–2. The amiloride-sensitive Na⁺ current was first measured when the high (70 mM) Na⁺ pipette solution included a mixture of GST fusion proteins containing the four individual WW domains of mouse Nedd4–2. We found that chord conductance of the amiloride-sensitive current was the same as that observed with the zero Na⁺ pipette solution (Fig. 2 ). This demonstrates that even when the WW domains of Nedd4–2 are present as separate proteins, they prevent down-regulation of ENaC by intracellular Na⁺. We then investigated whether mixtures containing only three of the four WW domains of Nedd4–2 were able to prevent inhibition of the amiloride-sensitive current by increased intracellular Na⁺. We found that when either a mixture of WW2, WW3, and WW4 or a mixture of WW1, WW3 and WW4 was present in the high Na⁺ pipette solution, the amiloride-sensitive current was identical to that measured with zero Na⁺ pipette solution (Fig. 2) . Mixtures of WW1, WW2, and WW3 or of WW1, WW2, and WW4, on the other hand, were unable to prevent the inhibition of the amiloride-sensitive current by the high Na⁺ pipette solution. The failure of the WW1, WW2, and WW3 mixture and of the WW1, WW2, and WW4 mixture to prevent Na⁺ feedback inhibition of the amiloride-sensitive current indicates that WW1 and WW2 are not sufficient to prevent interaction of ENaC with endogenous Nedd4 or Nedd4–2. It also indicates that neither WW3 nor WW4 alone is sufficient to prevent this interaction. On the other hand, our finding that the two mixtures that contain WW3 as well as WW4 prevent Na⁺ feedback inhibition of ENaC indicates that WW3 and WW4 are both required to prevent the interaction between the channel and Nedd4/Nedd4–2. This was confirmed when we included a mixture of just WW3 and WW4 in the high Na⁺ pipette solution and found it was sufficient to prevent inhibition of the amiloride-sensitive current (Fig. 2) . These studies thus indicated that WW3 and WW4 of Nedd4–2 must be present in the pipette solution to prevent down-regulation of ENaC by the endogenous Nedd4 or Nedd4–2. We had previously reported that in the case of mouse Nedd4, all three WW domains must be present in the pipette solution to prevent channel inhibition by high intracellular Na⁺. Given the high degree of homology of these proteins, this difference in behavior was so striking that we repeated some of the original experiments on Nedd4 to confirm them. We found that, as previously reported, a mixture of the two WW domains of mouse Nedd4 known to bind all three ENaC subunits in vitro (Nedd4 WW2 and Nedd4 WW3) did not prevent inhibition of the ENaC by the high Na⁺ pipette solution. A mixture of all three WW domains of Nedd4 was, however, able to prevent Na⁺ feedback regulation of the Na⁺ current (Fig. 2) .

View larger version (21K): [in this window] [in a new window]   Figure 2. The effects of Nedd4–2 or Nedd4 WW domains on the chord conductance of the amiloride-sensitive Na+ current. The whole-cell currents in salivary duct cells studied with 70 mM Na+ pipette solution plus a mixture of GST fusion proteins containing Nedd4–2 WW domains as follows. WW1–4 each at a concentration of 100 µg/mL; a mixture of WW2 + WW3 + WW4, or WW1 + WW3 + WW4, or WW1 + WW2 + WW3, or WW1 + WW2 + WW4 each at a concentration of 133 µg/mL; WW3 and WW4 each at a concentration of 200 µg/mL. Nedd4 WW2 and WW3 (at 200 µg/mL) or WW1–3 (each at 133 µg/mL) were used in the 70 mM Na+ pipette solution. The data are given as mean ± SE, with the number of experiments shown in parentheses. The dotted line indicates mean chord conductance of the amiloride-sensitive current observed with zero Na+ (n=4).

CONCLUSIONS AND SIGNIFICANCE

We have described here the identification of two alternatively spliced forms of Nedd4–2 that differ in 20 amino acid residues inserted just upstream of WW2. These spliced forms are expressed in a highly tissue-specific manner, suggesting they may have tissue/cell-specific functions. Note that the long Nedd4–2 isoform is expressed mostly in tissues that express ENaC, such as kidney, gut, lung, and salivary gland. However, in Xenopus oocytes, both isoforms are able to inhibit ENaC function similarly, suggesting that the presence of 20 amino acids in Nedd4–2 does not have a significant effect on ENaC regulation. We believe that although both Nedd4–2 isoforms appear to inhibit ENaC similarly, they may differentially regulate other undiscovered tissue-specific targets in vivo.

Our binding data indicate that WW3 and WW4 domains mediate the interaction between Nedd4–2 and ENaC subunits, whereas WW1 and WW2 do not show appreciable binding to any of the ENaC subunits (Fig. 1) . These data differ from binding data with Nedd4, where WW1 does not bind ENaC subunits but all other WW domains do, albeit with varying affinities. Our SPR data suggest that WW domain-ENaC binding/dissociation kinetics is rapid and that WW3 has the highest affinity for all three ENaC subunits. Our whole-cell patch clamp results show that the presence of the Nedd4–2 WW3 and WW4 is necessary and sufficient to prevent down-regulation of ENaC by high intracellular Na⁺ in ENaC-expressing salivary epithelial cells (Fig. 3 ). This implies that of the 4 WW domains in Nedd4–2, only WW3 and WW4 play a critical role in Na⁺ feedback regulation of ENaC in mammalian cells. The data also suggest that Nedd4–2 WW3 and WW4 interact with distinct noninterchangeable sites in ENaC. Our study is the first report of the role of individual Nedd4–2 WW domains in the regulation of endogenous mammalian ENaC in ENaC-expressing cells. In contrast to our results with Nedd4–2, studies of the closely related mouse Nedd4 protein show that it uses three WW domains to interact with ENaC. In the case of mouse Nedd4, all three domains are involved in this interaction; for human Nedd4, which has four WW domains, WW2, WW3, and WW4 are required. Our previous studies on mouse mandibular cells indicated that GST-WW domains from mouse Nedd4 selectively interact with three distinct sites and that since WW1 of Nedd4 does not bind ENaC, one of the sites is not on the Na⁺ channel. The findings for Nedd4 and Nedd4–2 can thus be reconciled if either Nedd4–2 WW3 or WW4 is able to indiscriminately interact with two of the three sites that are the targets of the WW domains of Nedd4.

View larger version (21K): [in this window] [in a new window]   Figure 3. A potential model of Nedd4–2-mediated ENaC regulation. Under high cytosolic Na+ conditions, Nedd4–2 binds with PY motifs in ENaC subunits via WW 3 and WW4. Our data suggest that WW3 and WW4 bind to distinct sites on ENaC subunits and that both domains are necessary for ENaC down-regulation. WW1 and WW2 may interact with other yet unknown protein(s).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0497fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0497fje

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