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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Pharmacology and Toxicology, University of Lausanne; 2Service of Cardiology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 3The Hospital for Sick Children, Toronto; and 4Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Submitted 20 September 2004 ; accepted in final form 11 November 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Nav1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Nav1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Nav1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Nav1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Nav channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Nav1.5; human ether-à-go-go-related gene
-subunit with an apparent molecular mass of 260 kDa associated with small ancillary 
-subunits of 35 kDa (12). The -subunit is the pore-forming protein and is sufficient for functional expression in heterologous expression systems. The human genome contains 10 genes encoding -subunits (Nav1.1–Nav1.9 and Nax), which are expressed mostly in excitable cells (6). Interestingly, cells such as neurons and cardiomyocytes may simultaneously express several members of the Nav family, and in most cases, the specific role played by these different isoforms is not clear. Importantly, abnormal function of Navs due to naturally occurring mutations in genes coding for Nav1.1, Nav1.2, Nav1.4, and Nav1.5 cause severe neurological and cardiac disorders in humans (22).
Thus far, little is known about the molecular determinants of trafficking, targeting, sorting, and internalization of Nav. Recently, two studies provided molecular evidence that Nav may be targets of ubiquitin-protein ligases of the Nedd4/Nedd4-like family (5, 31). At least nine genes coding for such Nedd4/Nedd4-like enzymes have been found in the human genome (11). Several of the members of this family are found in the nervous system, where they have been proposed to play a role in central nervous system development and axon guidance (20, 25). These proteins are ubiquitin-protein ligases (E3) comprising a C2 domain, two to four WW domains, and a catalytic Hect domain (7, 27). The C2 domain is involved in membrane targeting, the WW domains are protein-protein interaction modules involved in substrate recognition, and the Hect domain is responsible for catalytic ubiquitin ligase (E3) activity. Thus far, besides Nav, two other ion channels mainly expressed in epithelial cells, i.e., the epithelial Na+ channel (ENaC) and the Cl– channel ClC5, have been shown to be regulated by Nedd4-2 (2) and WWP2 (28), respectively. Both E3s belong to the family of Nedd4/Nedd4-like proteins (11). The current working model proposes that upon binding of Nedd4/Nedd4-like proteins to their target membrane ion channels, the latter are ubiquitinated and thereafter internalized and/or degraded. The interaction between the Nedd4/Nedd4-like proteins and their targets is mediated by WW domains of the E3s and a PY motif that, with the exception of Nav1.4 and Nav1.9, is found in the COOH termini of Navs and ENaCs (Fig. 1). The canonical PY motif is represented by the minimal sequence (L/P)PxY (17). In the case of ClC5, the sequence shown to be important for Nedd4/Nedd4-like protein regulation is very similar, i.e., PPLPPY (28). In addition, in recent reports (9, 16), researchers have presented evidence that a hydrophobic residue in position +3 after the Tyr (Tyr+3) of the PY motif is involved in the binding to the WW-domain pocket, hence forming an "extended" PY motif with the sequence (L/P)PxYxx
, with being a hydrophobic residue. Interestingly, such a sequence is present and conserved among 7 of the 10 Nav -subunits as well as in all three ENaC subunits (Fig. 1).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Antibodies. Rabbit serum against human Nav1.5 COOH terminus, raised against a glutathione S-transferase (GST) fusion protein comprising residues 1,978–2,016, was a gift from Alomone Laboratories (Jerusalem, Israel) and was characterized previously (31). SP19 anti-pan-Nav rabbit polyclonal antibody was obtained from Upstate (Waltham, MA), anti-ubiquitin monoclonal FK2 antibody was from Affiniti Research (Exeter, UK), and anti-Nedd4-1 and anti-Nedd4-2 antibodies were described previously (14, 30). Anti-T7 and anti-actin antibodies were obtained from Sigma (Buchs, Switzerland).
Transfection and homogenization of HEK-293 cells. HEK-293 cells, either nontransfected or stably expressing Nav1.5, were transiently transfected with Nedd4-2 constructs using Ca2+-phosphate. Two days after transfection, cells were solubilized by 30-min rotation at 4°C in lysis buffer containing 20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, and Complete protease inhibitor cocktail (1 tablet/25 ml; Roche, Rotkreuz, Switzerland). Soluble fractions were recovered in supernatants after 15 min of centrifugation at 18,000 g. Protein content was measured using Bradford test-based CooAssay reagent (Uptima, Basel, Switzerland) with BSA as a reference.
Brain tissue preparation. Brains of 3- to 4-month-old mice (C57BL/6 strain, bred in-house) were excised and transferred to lysis buffer containing (in mM) 20 HEPES, pH 7.6, 125 NaCl, 10% glycerol, 1 EGTA, 1 EDTA, 1 dithiothreitol (DTT), 1 PMSF, and Complete protease inhibitor cocktail (Roche). Tissue was homogenized using a Polytron and a Teflon homogenizer. Triton X-100 was added to a final concentration of 1%, and solubilization was induced by rotating for 1 h at 4°C. The soluble fraction from 15-min centrifugation at 13,000 g (4°C) was used as a source of Nedd4-2 for pull-down assays. Animal experiments were performed in accordance with Swiss law.
Pull-down assays. The cDNA encoding the last 57, 56, and 66 amino acids of rat Nav1.2, human Nav1.3, and Nav1.5, respectively, were cloned into pGEX-4T1 (Amersham, Otelfingen, Switzerland). In addition, we generated Tyr-to-Ala PY-motif mutants of each construct. Expression of GST fusion proteins in Escherichia coli cells was induced with 0.2 mM isopropylthiogalactoside for 3 h at 30°C. Cells were harvested by centrifugation and resuspended in lysis buffer. Soluble fractions from a 15-min centrifugation at 13,000 g (4°C) were rotated for 1 h in the presence of glutathione (GSH)-Sepharose at 4°C. Beads containing bound fusion proteins were recovered after washing and used in pull-down experiments. Amounts (2 µg) of GST fusion proteins used in the pull-downs were verified by fluorescent staining of proteins in gels using the Insite dye (National Diagnostics, Basel, Switzerland). GST pull-down assays of soluble fractions of brain lysate were performed using GSH-Sepharose beads containing either GST or one of the GST fusion proteins. After overnight incubation and washing of the beads (in mM: 20 HEPES, pH 7.6, 500 NaCl, 1% Triton X-100, and 1 PMSF), bound Nedd4-2 was detected using Western blot analysis.
Detection of ubiquitinated Nav1.5. To study the ubiquitination of Nav1.5 by Nedd4-like proteins, HEK-293 cells were transfected with either empty vector or wild-type (WT) or mutant Nav alone or together with Nedd4-2, Nedd4-1, or T7-tagged WWP2. Cells were solubilized as described previously in lysis buffer supplemented with 10 mM N-ethylmaleimide. Samples (1 ml) of soluble fractions containing 1 mg of protein were incubated for 2 h under rotation at 4°C in the presence of 2 µl of anti-Nav1.5 serum. After the addition of 25 µl of protein A-Sepharose (drained volume), the protein solutions were incubated for an additional 1 h as before. After extensive washing, bound proteins were eluted by 5 min of boiling of the beads in 50 µl of sample buffer containing 50 mM DTT, and ubiquitination levels were determined by performing Western blot analysis using FK2 antibody.
Kd measurement. Peptides representing sequences of human Nav1.5 and human ether-à-go-go-related gene (hERG) were synthesized by the Hospital for Sick Children/Advances Protein Technology Centre (Toronto, ON, Canada). The mass and purity of the peptides were confirmed by performing electrospray mass spectrometry. Peptide sequences were human Nav1.5, STSFPPSYDSVTR, and hERG, QRMTLVPPAYSAVTT. Lyophilized peptides were resuspended in 150 mM KCl and 10 mM K+-phosphate, pH 6.5. Peptide concentrations were measured in 6.0 M guanidine HCl at Ala280 (26). The WW domains of Xenopus Nedd4-2 (GenBank accession no. CAA03915), WW1 (residues 186–225), WW2 (residues 377–416), WW3 (residues 489–528), and WW4 (residues 540–579) were subcloned into pQE-30 and expressed as NH2-terminal MRGS (methionine, arginine, glycine, serine)-His6-tagged proteins. These WW domains are identical to the human Nedd4-2 WW domains. Proteins were expressed and purified from E. coli M15 pREP4 as described previously (9). Intrinsic tryptophan fluorescence of the WW domains was used to monitor peptide binding. Fluorescence measurements were obtained using a Hitachi F-2500 fluorescence spectrophotometer at 25°C with excitation and emission wavelengths of 298 and 333 nm, respectively, and slit width of 2.5 nm. Experiments were measured in 150 mM KCl and 10 mM K+-phosphate, pH 6.5, with WW-domain concentrations kept constant at 2 µM. Peptides were added at concentrations ranging from 0 to 1.2 mM. Calculations of the equilibrium dissociation constant (Kd) were performed as described previously (9, 16).
Electrophysiology.
For electrophysiological studies, HEK-293 cells were transiently cotransfected with 0.3 µg of Nav1.2, Nav1.5-WT, or mutant constructs and 1.4 µg of Nedd4-2, Nedd4-1, WWP2-WT, or mutant constructs or empty vector (control). HEK-293 cells stably expressing Nav1.3-WT were transiently transfected with either Nedd4-2-WT or CS-mutated Nedd4-2 (i.e., Nedd4-2 in which Cys801 was mutated into a Ser) cDNA (1.4 µg) or empty vector. Nav -subunits were not cotransfected. All transfections included 0.8 µg of cDNA encoding CD8 antigen as a reporter gene. Cells were incubated for 18 h with the transfection Ca2+-phosphate mix. After 24 h, cells were split at low density. Anti-CD8 beads (Dynal, Oslo, Norway) were used to identify transfected cells, and only decorated cells were analyzed.
Whole cell currents were measured at room temperature (22–23°C). The internal pipette solution was composed of (in mM) 60 CsCl, 70 Cs-aspartate, 1 CaCl2, 1 MgCl2, 10 HEPES, 11 EGTA, and 5 Na2-ATP, pH 7.2, with CsOH. The external solution contained (in mM) 130 NaCl, 5 CsCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, and 5 glucose, pH 7.4, with CsOH. Measurements were made using pClamp software, version 8 (Axon Instruments, Union City, CA) and a VE-2 amplifier (Alembic Instruments, Montreal, QC, Canada). Data were analyzed using pClamp software, version 8 (Axon Instruments), and KaleidaGraph software (Synergy Software, Reading, PA). Peak currents were measured using a current-voltage protocol, and Na+ current (INa) densities (expressed as pA/pF) were obtained by dividing the peak current by the cell capacitance obtained using the pClamp function.
Confocal imaging. HEK-293 cells were transiently transfected with 0.025 µg of a Nav1.5-yellow fluorescent protein (YFP) construct (33) (kind gift from Dr. T. Zimmer, University of Jena, Jena, Germany) and with Nedd4-2-WT or Nedd4-2-CS mutant (1.4 µg). Alternatively, to ascertain that the analyzed cells were transfected by both constructs, Nedd4-2-WT and -CS constructs that were tagged with green fluorescent protein (GFP) at the NH2 terminus by subcloning Nedd4-2 into peGFP-C1 (Clontech) were also transfected. We observed no difference between the Nedd4-2 GFP-tagged and nontagged constructs. For these experiments, we had to reduce 10-fold the amount of transfected Nav1.5 DNA compared with standard transfections, because under the latter conditions, the localization of the protein was restricted mainly to intracellular compartments (33). Two days after transfection, fluorescent proteins were visualized using confocal microscopy (LSM 510; Zeiss, Göttingen, Germany) with living cells. Optical sections were obtained at 512 x 1024-pixel resolution, and fluorescence intensities were analyzed using LSM software (Zeiss).
Statistical analysis. Data are represented as means ± SE. A two-tailed Student's t-test was used to compare the means.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the present study, we extended our investigations to two other Nav members expressed in the nervous system that have a PY motif identical to that of Nav1.5 (Fig. 1). For this purpose, we generated GST fusion proteins of the COOH termini of rat Nav1.2, human Nav1.3, and Nav1.5, comprising their PY motifs, as well as mutant forms in which the Tyr of the PY motif was mutated into Ala. We performed pull-down experiments on mouse brain lysates using these fusion proteins, and as presented in Fig. 2A, at least two variants of Nedd4-2 expressed in mouse brain were pulled down in a PY-motif-dependent manner. Nedd4-2 was recovered to a similar extent with all three fusion proteins. These results are similar to those obtained with Nav1.5 fusion proteins used in pull-down experiments using cardiac lysates (31). The presence of different sizes of Nedd4-2 in mouse brain may represent splice variants as recently reported for other tissues (13).
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80–90%. This downregulation required the ubiquitin ligase activity of the enzyme because catalytically inactive Nedd4-2 (Nedd4-2-CS) did not reduce Nav1.2 and Nav1.3 INa. In the case of Nav1.3, Nedd4-2-CS increased INa threefold, suggesting that the cell surface density of this channel isoform was regulated by an endogenous Nedd4-like activity. Similar results were obtained when studying the effect of Nedd4-2-CS on Nav1.5 and ENaC expressed in Xenopus oocytes (1, 2), suggesting that the catalytically inactive Nedd4-2 may, in particular conditions, exert an antagonistic effect on endogenous Nedd4-like proteins. Analysis of the extended PY motif of Nav1.5 and its interaction with Nedd4-like proteins. In recent studies (9, 16), the affinities of the PY motifs of the three ENaC subunits for Nedd4 WW domains were determined by measuring the change in intrinsic tryptophan fluorescence of the WW domains upon binding of peptides. To determine the binding affinity of the PY motif of Nav1.5 to WW domains of Nedd4, we performed similar experiments using a peptide comprising the PY motif of human Nav1.5 (1970STSFPPSYDSVTR1982). To asses whether the presence of a consensus PY motif is sufficient for binding of a peptide to a WW domain, we also tested another PY-motif-containing peptide found in the COOH-terminal region of the cardiac delayed rectifier hERG channel (voltage-gated K+ channel 11.1). The sequence of this peptide is 1069QRMTLVPPAYSAVTT1083.
The Nav1.5 PY-motif peptide was able to bind to the fourth WW domain (WW4) of Nedd4-2 with moderate affinity (55 µM), while its binding to each of the other three WW domains of Nedd4-2 was either very poor or undetectable (Table 1). This includes WW3, which was previously demonstrated to strongly bind the PY motif of -ENaC (Ref. 9 and Table 1). WW4 of rat Nedd4-1 bound the Nav1.5 peptide with somewhat lower affinity than the Nedd4-2 WW4 domain (Table 1), even though they share a high degree of sequence similarity. In contrast to the Nav1.5 PY peptide, the PY peptide derived from the hERG channel bound very poorly to the WW domains of Nedd4-2 (Table 1), suggesting that subtle differences in the peptide sequence, besides the PPxYxxV consensus residues, can play an important role in these interactions. Moreover, the latter results are consistent with pull-down experiments that showed that GST-fusion proteins comprising the PY motif of hERG did not bind Nedd4-2 (Gavillet B and Abriel H, unpublished data).
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-ENaC PY motif (9, 16). To further investigate this possibility, we performed an alanine scan of the region encompassing the putative extended PY motif of Nav1.5 by replacing all single residues between Pro1974 and Val1980 with Ala. With the use of structural analysis, the homologous region of ENaC was previously shown to be involved in binding to the WW domains (16). These point mutations were generated both in the DNA constructs used for the production of GST COOH-terminal fusion proteins and for the expression of full-length Nav1.5 proteins in HEK-293 cells. Figure 3A illustrates the results of the pull-down experiments performed with these mutant PY-motif fusion proteins using lysates of HEK-293 cells transiently transfected with Nedd4-2, Nedd4-1, or WWP2. These three proteins are members of the family of Nedd4-like protein-ubiquitin ligases (27) and have been shown to be expressed in cardiac tissues as well as in the nervous system. The results of these pull-down experiments indicate that the sequence requirements for binding of all three Nedd4-like proteins are very similar (Fig. 3A) as illustrated in the quantification of three such experiments (Fig. 3B). Replacement of Nav1.5 Pro1974, Pro1975, and Tyr1977 with Ala strongly reduced the amount of bound Nedd4-like proteins. Interestingly, and in accordance with previous results obtained in our laboratory (31), replacement of the Val1980 with an Ala (also a hydrophobic residue) did not reduce the binding of any of the three ubiquitin ligases. Indeed, investigators at our laboratory (31) previously found that only the substitution of Val1980 with charged residues altered the binding of Nedd4-2 to the Nav1.5 COOH terminus under pull-down conditions. An estimation of the relative binding affinity of the three E3s for the WT PY motif of Nav1.5 can be obtained by comparing the amount of bound E3 with the quantity of the same E3 present in the cell lysate (Fig. 3A). Consistent with the Kd values that we obtained experimentally (Table 1), this ratio is in a range similar to that found for Nedd4-2 and Nedd4-1 as well as WWP2.
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Role of other ubiquitin-protein ligases of the Nedd4/Nedd4-like family. As presented above, we found that besides Nedd4-2, Nedd4-1 and WWP2 also bind to the COOH-terminal segment of Nav1.5 in a PY-motif-dependent manner (Fig. 3, A and B). We therefore tested whether these other Nedd4-like E3s may ubiquitinate Nav1.5 as previously described for Nedd4-2 in HEK-293 cells (31). Figure 4A shows that, by using an anti-ubiquitin antibody, ubiquitination of immunoprecipitated Nav1.5 from the total cellular pool was clearly apparent when Nedd4-2 was coexpressed. In contrast, a much weaker increase in the ubiquitin signal was observed using Nedd4-1 and WWP2, despite comparable expression levels. Consistent with our functional and binding experiments, Nav1.5-Y1977A mutant channels were not ubiquitinated by any of the three tested E3s (Fig. 4A).
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30%. These results suggest that either differences in binding affinity (e.g., between Nedd4-2 WW4 and Nedd4-1 WW4 domains; Table 1) or factors other than binding to the PY motif are responsible for the disparity in the efficiency with which Nedd4-like enzymes mediate ubiquitination and downregulation of Nav1.5. These findings raise the possibility that, assuming proper cellular localization in cardiac cells, Nedd4-1 and WWP2 could antagonize Nedd4-2 action. This hypothesis was tested by cotransfecting, with Nav1.5 and Nedd4-2, a twofold excess of cDNA encoding either Nedd4-1 or WWP2 compared with that of Nedd4-2. Western blots shown in Fig. 4E illustrate that, with this protocol, we approximately doubled the amount of Nedd4-1 and WWP2 compared with the conditions shown in Fig. 4, B and C. Under these conditions, WWP2 partially inhibited the Nedd4-2-dependent downregulation of Nav1.5 INa (Fig. 4D). This effect was not mediated by a WWP2-dependent downregulation of Nedd4-2, because Nedd4-2 levels did not change upon cotransfection of the other E3 (Fig. 4E). In contrast, cotransfected Nedd4-1 did not interfere with Nedd4-2-dependent INa downregulation (Fig. 4D), despite its capacity to bind at the Nav1.5 COOH terminus as shown in Fig. 3A.
Cellular mechanisms of Nedd4-2-mediated downregulation of Nav1.5 INa.
Xenopus Nedd4, the homolog of human Nedd4-2 (14), has been shown to decrease the cell membrane density of ENaC expressed in Xenopus oocytes (2). In the case of Nav1.5, it was previously shown that the biophysical properties of Nav1.5 remaining at the cell surface were not altered upon Nedd4-2 coexpression and that the peripheral localization of Nav1.5 in HEK-293 cells was lost in the presence of Nedd4-2 (31). Together, these findings suggested that the Nedd4-2-dependent reduction in Nav1.5 INa is caused by a decreased density of the channels at the cell membrane. Figure 5A shows the difference in cellular localization of a YFP-tagged form of Nav1.5 with confocal microscopic imaging of transiently transfected HEK-293 cells with or without Nedd4-2-WT. Upon transfection of Nedd4-2-CS, the Nav1.5 cellular localization was similar to that observed under control conditions (Fig. 5A). Quantification of the fluorescence intensity in a region of 0.75 µm encompassing the cell-cell contact region shows a decrease of 80% (Fig. 5B) in Nedd4-2-WT-cotransfected cells. No significant decrease was observed with Nedd4-2-CS. These data strongly suggest that the observed 80% reduction of INa (Fig. 3C) is caused by a reduced density of Nav1.5 at the cell surface.
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44 h posttransfection (t = 20 h in Fig. 6A, thin biphasic curve). Afterward, INa recovered gradually, reaching values close to those of the control transfected cells (72 h after transfection). This phenomenon is most probably caused by the parallel increase and decrease in Nedd4-2 expression over time as revealed in Western blot experiments (Fig. 6, C and D). When BFA was added to the Nedd4-2-transfected cells, INa decayed more rapidly than in control transfected cells treated only with BFA. Monoexponential fit (Fig. 6, A and B, dotted curves) of the averaged data yielded a decay half-time of 5.8 h. These findings are consistent with a model in which Nedd4-2 accelerates the rate of internalization of Nav1.5 channels from the cell surface. A more complex model in which Nedd4-2 could also act simultaneously on the secretory pathway cannot be excluded on the basis of these data. On the other hand, it cannot be excluded that BFA disrupts unknown factors involved in stabilizing Nav1.5 at the cell membrane.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Regulation of Nav channels by Nedd4-like E3s can be extended to neuronal channels.
The conservation of the extended PY motif with the consensus sequence (L/P)PxYxx(V/L) in the COOH-terminal region of seven Nav members is a striking feature. The sequences are identical (Fig. 1), with the exception of leucine instead of the first proline in Nav1.6. In fact, it has been reported that the first proline in PY motifs can be substituted with leucine without loss of binding affinity to WW domains (17). The results of the present work, along with those of Fotia et al. (5), clearly have shown that not only the cardiac Nav1.5 channels but also probably most of the neuronal channels can be regulated by Nedd4-like proteins. Our observations indicate that common features allowing for ubiquitination and internalization, other than the PY motif, are also shared by these different channels. At present, very little is known about the molecular and cellular mechanisms involved in the regulation of the membrane density of Nav channels in excitable cells. As exemplified by a number of human diseases caused by sometime subtle gain- or loss-of-function mutations in Nav genes, normal cellular function does not tolerate variation in the number of functional Nav channels at the cell surface. Thus far, no mutation in the PY motif of any Nav genes has been described. Besides the probable role of the -subunits in increasing the channel density at the cell membrane (12), the present work further supports the role played by Nedd4-like E3s in regulating the density of Nav channels.
The ubiquitin system, and in particular this class of E3s, has been shown to play an important role in the nervous system (8). Nedd4-1, the first identified member of this family of nine genes (11), is strongly expressed in the embryonic mouse brain (20, 21), and its Drosophila homolog, dNedd4, is an important factor in axonal guidance during brain development by targeting the protein Commissureless (25). Besides neurological diseases such as genetic forms of epilepsy caused by mutations in Nav1.1 and Nav1.2 genes (22), Nav most probably play a central role in acquired disorders such as chronic pain and multiple sclerosis (32). It can be speculated that the Nedd4-like-dependent regulation or dysregulation of Navs may represent a mechanism involved in normal and pathological states.
Molecular determinants of the interaction between Nav and Nedd4-like proteins.
The structural factors important for the interaction of the -ENaC extended PY motif, PPnYdsL, with the WW domains of Nedd4-1 and Nedd4-2 have been studied in detail (9, 15, 16). The WW domains form a hydrophobic binding surface comprising two stabilizing surfaces promoting the interaction with the PY-motif ligand. An XP groove surface interacts with the polyproline type II helix, and a second surface interacts with the Tyr of the PY motif. These studies also provided strong evidence that the residues following the traditional PPxY motif make a sharp helical turn, allowing the methyl group of the Tyr+3 aliphatic residue (Leu621 in -ENaC and Val1980 in Nav1.5) to interact with the WW domain, thus providing additional binding energy. Overall, the binding and functional data presented in the present report as well as in a previous study conducted in our laboratory (31) are in agreement with a similar type of interaction that takes place between the extended Nav PY motif and the WW domains of Nedd4-2 or other Nedd4-like proteins. The measured affinity of Nav1.5 PY-motif peptide with WW domains was highest in the WW4 of Nedd4-2, a finding that contradicts that of Fotia et al. (5), who found that the interaction was stronger with the WW3 of Nedd4-2, although Kd measurements were not provided in their study. This inconsistency may be caused by the fact that the binding assays were different (tryptophan fluorescence measurement vs. Far Western blot analysis). The absolute value of 55 µM represents a moderate-affinity interaction. However, as suggested by the specificity of our findings, it is possible that in the cellular context, other factors such as partner proteins may increase the strength of this interaction. Nevertheless, this interaction seems to be weak and transient as exemplified by our failure to coimmunoprecipitate the two full-length proteins from cell lysates (van Bemmelen MX and Abriel H, unpublished data).
The results of the Nav1.5 PY-motif alanine scan showed a slight discrepancy between the binding (pull-down) experiments and the Nedd4-2-dependent downregulation of the Nav1.5 currents. Single replacements of the Tyr+1 to Tyr+3 residues (i.e., Asp-Ser-Val) with Ala did not interfere with the capacity of the GST fusion proteins to interact with the tested E3s. In contrast, the channels bearing the same mutations expressed in HEK-293 cells were less efficiently regulated by Nedd4-2 compared with WT Nav1.5 (Fig. 3C). This observation suggests either that the pull-down approach used does not have enough resolution to discriminate low-affinity differences or that the functional consequences of the interaction between Nedd4-2 and Nav1.5 may be dependent on the integrity of this sequence in the cellular context.
Surprisingly, we did not observe any saturable binding to any WW domains using the hERG PY-motif peptide, despite the fact that its sequence (PPaYsaV) corresponds well to the predicted structural requirements. One possible difference may be the Tyr+1 residue that is occupied by a negatively charged residue in the PY-motif sequences with the highest affinities (-ENaC and Nav). Interestingly, in a recent large-scale screen of WW domains binding peptides (10), peptides with negative charges in positions Tyr+1 and +2 were shown to be preferred. Further experiments are necessary to elucidate the mechanisms underlying this observation.
Diversity in the E3s and specificity in the effects. In the current study, we tested three of the nine known E3s present in the human genome, i.e., Nedd4-1, Nedd4-2, and WWP2. Analogously to the work of Fotia et al. (5), who tested Nedd4-1 and Nedd4-2, we observed striking differences in the capacity of these ligases to ubiquitinate and regulate Nav channels. Indeed, despite the fact that Nedd4-1 and WWP2 were able to bind well to the PY motif of Nav1.5, their efficacy in ubiquitinating and downregulating Nav1.5 was very weak compared with Nedd4-2. Furthermore, we observed that, when coexpressed with Nedd4-2, WWP2 was competing with the Nedd4-2-dependent downregulation of Nav1.5 currents. The mechanism of such competition, described in the present report for the first time, is not clear. It seems, however, that it cannot be based solely on the fact that both E3s compete for the same binding site, i.e., PY motif of Nav1.5, because such competition was not observed with Nedd4-1 (Fig. 4D). It could be that unknown cellular factors are necessary for both Nedd4-2 and WWP2 and that coexpression of WWP2 may reduce the availability of these factors for Nedd4-2. Together, these results illustrate the potential complexity of these regulatory mechanisms, and, as a consequence, further investigations are needed to answer these questions.
Cellular mechanisms of Nedd4-2 regulation.
In this study, we have provided direct experimental evidence for a role of Nedd4-2 in internalization of cell membrane ion channels in mammalian cells. Coexpression of Nedd4-2 with Nav1.5 leads to a strong redistribution of Nav1.5 from the cell surface to undefined intracellular compartments. In agreement with these findings, the experiments performed in the presence of BFA indicated that Nedd4-2 increased the Nav1.5 disappearance rate. Mutations of the PY motifs of ENaC - or 
-subunits found in humans lead to a hereditary form of hypertension known as Liddle's syndrome. It has been demonstrated clearly that ENaC in patients with Liddle's syndrome are less efficiently regulated by Nedd4-like proteins and also that they accumulate at the cell surface of Xenopus oocytes (2, 18) and renal cells (3). When BFA was used to block the cellular secretory pathway, Nedd4-2 more than doubled the rate at which the INa decreased. This observation further supports the model proposing that Nedd4-2 directly ubiquitinates Nav1.5 as presented in Fig. 4A and that this ubiquitination enhances the rate of endocytosis of the channels. However, thus far, the molecular mechanisms underlying this phenomenon are poorly understood, and analogously to recent reports (24, 29), ubiquitination of Nav1.5 may be important for proper sorting at the early endosomal stage rather than for the internalization process. Increasing the sorting of Nav1.5 toward early endosomes and/or decreasing the putative recycling of channels to the membrane could result in fewer channels at the cell surface.
Physiological relevance.
At this stage, the relevance of these findings in normal and abnormal cellular physiology is only speculative. Several observations, however, point to a physiological role of this proposed mode of regulation of Nav channels. The extended PY motif found in Nav channels is very similar to motifs found in the different subunits of ENaC, most particularly in the -subunit. This motif is very well conserved among the different members of the family of Navs, despite the fact that the distal parts of the COOH termini of these channels are variable. Moreover, ubiquitinated forms of Nav1.5 have been found in cardiac tissues, suggesting a physiological role for this type of protein posttranslational modification (31). Finally, researchers in this area face a rather complex situation, because there is, on the one hand, a family with seven channels containing a PY motif, and on the other hand, a family of nine Nedd4/Nedd4-like ubiquitin-protein ligases that are widely expressed in excitable cells. In theory, this allows the possibility of a very large number of complex ways to regulate Nav density at the cell membrane. Most likely, only systematic and large-scale approaches using small interfering RNA silencing cellular models, total knockout mice, and tissue-specific knockout mice may provide information that will help to address the issue of complexity.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* J.-S. Rougier and M. X. van Bemmelen contributed equally to this work. 
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), cells treated with 50 ng/ml brefeldin A (BFA) (
), transiently transfected 24 h before addition of BFA (t = 0) with Nedd4-2 (
), and transiently transfected 24 h earlier with Nedd4-2 and treated with BFA (t = 0) (
). The bold and dotted curves are monoexponential fits of the INa decays after BFA treatment of control (bold curve) and Nedd4-2-transfected cells (dotted curve). n = 4–7 cells per condition. The thin curves were obtained via interpolation of the data points (see text for details). B: normalization of the fitted INa decays shown in A after BFA suggests that Nedd4-2 increases the internalization rate of Nav1.5 (bold line, BFA alone; dotted line, BFA+Nedd4-2). C and D: as shown in A (
) correlated with the observed negative but transient effect of Nedd4-2 on the Nav1.5-mediated currents (D,