Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)*

Background: The hERG K+ channel plays an important role in the repolarization of cardiomyocytes where Rab4 is present. Results: Overexpression of Rab4 decreases the density of mature hERG channels. This effect is mediated through enhanced expression of the ubiquitin ligase Nedd4-2. Conclusion: Rab4 regulates hERG channel density via Nedd4-2. Significance: Our data revealed a novel pathway for Nedd4-2 and hERG regulation. The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit of the rapidly activating delayed rectifier K+ channel in the heart, which plays a critical role in cardiac action potential repolarization. Dysfunction of IKr causes long QT syndrome, a cardiac electrical disorder that predisposes affected individuals to fatal arrhythmias and sudden death. The homeostasis of hERG channels in the plasma membrane depends on a balance between protein synthesis and degradation. Our recent data indicate that hERG channels undergo enhanced endocytic degradation under low potassium (hypokalemia) conditions. The GTPase Rab4 is known to mediate rapid recycling of various internalized proteins to the plasma membrane. In the present study, we investigated the effect of Rab4 on the expression level of hERG channels. Our data revealed that overexpression of Rab4 decreases the expression level of hERG in the plasma membrane. Rab4 does not affect the expression level of the Kv1.5 or EAG K+ channels. Mechanistically, our data demonstrate that overexpression of Rab4 increases the expression level of endogenous Nedd4-2, a ubiquitin ligase that targets hERG but not Kv1.5 or EAG channels for ubiquitination and degradation. Nedd4-2 undergoes self- ubiquitination and degradation. Rab4 interferes with Nedd4-2 degradation, resulting in an increased expression level of Nedd4-2, which targets hERG. In summary, the present study demonstrates a novel pathway for hERG regulation; Rab4 decreases the hERG density at the plasma membrane by increasing the endogenous Nedd4-2 expression.

The human ether-a-go-go-related gene (hERG) 2 encodes the pore-forming subunit of the rapidly activating delayed rectifier K ϩ channel (I Kr ) in the heart, which plays an important role in cardiac action potential repolarization (1,2). A reduction or an increase in I Kr can lead to long or short QT syndrome, both of which predispose affected individuals to fatal arrhythmias and sudden cardiac death (3,4). Dysfunction of hERG channels can be caused by drugs that interfere with the channel gating. In addition, factors such as hypokalemia (a reduced plasma K ϩ concentration) and certain drugs such as pentamidine and probucol impair hERG function by decreasing the expression level of the hERG channel (5)(6)(7).
The homeostasis of hERG protein at the plasma membrane is a balance of anterograde and retrograde trafficking mechanisms. The recycling of internalized protein to the plasma membrane also plays an important role in this process. The hERG channel is initially synthesized in the endoplasmic reticulum as the immature core-glycosylated form with a molecular mass of 135 kDa. The immature hERG channel then undergoes full glycosylation in the Golgi apparatus to become the mature form with a molecular mass of 155 kDa, which is transported to the plasma membrane as functional channels (8). Our previous works have shown that ubiquitination of hERG protein at the plasma membrane triggers internalization of hERG channels under low K ϩ conditions (5,9,10). Furthermore, the ubiquitin (Ub) ligase Nedd4-2 ubiquitinates and degrades mature hERG channels (11)(12)(13). However, whether the internalized hERG proteins can be recycled back to the plasma membrane remains to be determined.
Ion channels are targets of small GTPases (14) which play various roles in the regulation of protein trafficking (15)(16)(17). It has been shown that Rab1 and Rab2 regulate endoplasmic retic-* This work was supported by the Canadian Institutes of Health Research ulum-to-Golgi transport (18,19); Rab4 is localized at the early sorting endosome and is responsible for rapid/direct recycling from early endosomes to the cell surface (20,21); Rab5 regulates the fusion between endocytic vesicles and early endosomes (22,23); Rab7 has primarily been implicated in the transport from early to late endosomes and plays an essential role in the maintenance of the perinuclear lysosome compartment (17,22,24); and Rab11 is involved in mediating slow recycling from endosomes to the plasma membrane (25).
In the present study, we investigated the regulatory effects of various Rabs on hERG channels. Our data unexpectedly revealed that Rab4 significantly decreases the expression level of hERG at the plasma membrane. Mechanistically, we found that Rab4 decreases the ubiquitination of Nedd4-2, which results in an increase in Nedd4-2 expression. The increased Nedd4-2 then decreases hERG expression at the plasma membrane by targeting the PY motif in the C terminus of hERG channels.

EXPERIMENTAL PROCEDURES
Molecular Biology-hERG cDNA was provided by Dr. Gail Robertson (University of Wisconsin-Madison); a hERG-expressing human embryonic kidney (HEK) 293 stable cell line (hERG-HEK cells) was provided by Dr. Craig January (University of Wisconsin-Madison). The human ether-a-go-go (EAG) cDNA was provided by Dr. Luis Pardo (Max-Planck Institute of Experimental Medicine, Göttingen, Germany); Kv1.5 cDNA (encoding the cardiac ultrarapidly activating delayed rectifier K ϩ channel) was provided by Dr. Michael Tamkun (Colorado State University, Fort Collins, CO). GFP-tagged Rab1, Rab4, inactive Rab4 mutant Rab4N121I, Rab5, Rab7, and Rab11 plasmids were obtained from Addgene and Dr. Terry Hébert (McGill University, Montreal, ON, Canada). The scrambled control siRNA and Rab4A siRNA targeting human Rab4 were purchased from Santa Cruz Biotechnology. The Rab4A siRNA targeting rat Rab4 was purchased from Sigma-Aldrich. The human Nedd4-2 plasmid in pBluescript II was obtained from Kazusa DNA Research Institute (Chiba, Japan). The open reading frame of Nedd4-2 was amplified using polymerase chain reaction (PCR) and cloned into HA-pcDNA3 (Invitrogen) to generate HA-tagged Nedd4-2. The plasmid of the catalytically inactive form of Nedd4-2, Nedd4-2-C801S mutant (Nedd4-2CS), was provided by Dr. Hugues Abriel (University of Bern, Switzerland). To disrupt the Nedd4-2 interaction with hERG, we generated the hERG point mutation Y1078A and C-terminal truncation mutation ⌬1073 using PfuUltra Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA). The mutations were confirmed by DNA sequencing (Eurofins MWG Operon, Huntsville, AL). Stable cell lines were created and maintained at 37°C in minimum essential medium (Life Technology) supplemented with 10% fetal bovine serum (FBS) and 0.4 mg/ml G418 (Invitrogen). For transient transfection, 2 g of the plasmid of interest was transfected into hERG-HEK or HEK 293 cells grown in a 35-mm dish at 60 -70% confluence using Lipofectamine 2000 (Invitrogen). A green fluorescent protein (GFP) plasmid (0.5 g, pIRES2-EGFP; Clontech) was co-expressed to identify transfected cells for electrophysiological studies. After transfection, cells were cultured in 10% FBS-supplemented minimum essential medium for 24 h before experiments.
Neonatal Rat Ventricular Myocyte Isolation-Experimental protocols used for animal studies were approved by the Animal Care Committee of Queen's University. Single cardiomyocytes were isolated from 1-day-old Sprague-Dawley rats of either sex using enzymatic dissociation methods as described previously (7). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Invitrogen) with 10% FBS. Cardiomyocytes were grown on glass coverslips for electrophysiological and immunocytochemical studies and in 60-mm dishes for Western blot analysis.
Electrophysiological Recordings-For recording the activities of WT and mutant hERG, as well as Kv1.5 and EAG channels stably expressed in HEK 293 cells, the whole-cell patch clamp method was used. The bath solution contained 135 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES (pH 7.4). The pipette solution contained 135 mM KCl, 5 mM EGTA, 1 mM MgCl 2 , and 10 mM HEPES (pH 7.2). The hERG current (I hERG ), EAG current (I EAG ), or Kv1.5 current (I Kv1.5 ) from respective stable HEK cell lines was recorded by depolarizing steps to voltages between Ϫ70 mV and ϩ70 mV in 10-mV increments. The tail currents were recorded upon a repolarizing step to Ϫ50 mV. The holding potential was Ϫ80 mV. For the current amplitude analysis, the peak tail current at Ϫ50 mV following the 50-mV depolarizing step for the hERG channel, and the pulse current at the end of the depolarizing step to 50 mV for the Kv1.5 and EAG channels were used. For the recording of native I Kr in cultured neonatal rat cardiomyocytes, the pipette solution contained 135 mM CsCl, 5 mM MgATP, 10 mM EGTA, and 10 mM HEPES with pH 7.2 by CsOH. The bath solution contained 135 mM CsCl, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, and 10 M nifedipine with pH 7.4 by CsOH. The current was evoked by depolarizing cells to voltages between Ϫ70 mV and 70 mV in 10-mV increments. The current amplitude upon repolarization to the Ϫ80 mV holding potential after the depolarizing step of 50 mV was used to measure the amplitude of native I Kr (26). Patch clamp experiments were performed at room temperature (22 Ϯ 1°C).
Western Blot Analysis and Co-immunoprecipitation-Following treatments, hERG-HEK cells were cultured in 35-mm dishes for 24 h. Whole-cell proteins were extracted and separated on 8% or 15% SDS-PAGE gels, transferred onto PVDF membranes, and blocked for 1 h with 5% nonfat milk. The membranes were incubated with appropriate primary antibodies for 1 h at room temperature and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies. Actin expression was used for the loading control. The blots were visualized with Fuji film using the ECL detection kit (GE Healthcare). To quantify the Western blot data, the band intensities of proteins of interest in each gel are first normalized to their respective actin intensities; the normalized intensities are then compared with the band intensities from control cells, and are expressed as relative values to their controls.
For co-immunoprecipitation, whole-cell proteins (0.5 mg) were incubated with the appropriate primary antibody overnight at 4°C and then precipitated with protein A/G plus aga-rose beads (Santa Cruz Biotechnology) for 4 h at 4°C. The beads were washed three times with ice-cold radioimmunoprecipitation assay lysis buffer, resuspended in 2 ϫ Laemmli sample buffer, and boiled for 5 min. The samples were centrifuged at 20,000 ϫ g for 5 min. The supernatants were collected and analyzed using Western blot analysis.
Immunofluorescence Microscopy-hERG-HEK cells were transfected with GFP, GFP-tagged Rab4, or GFP-tagged Rab4N121I. Twenty-four hours after transfection, the cells were fixed with freshly prepared 4% paraformaldehyde for 15 min. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% bovine serum albumin (BSA) for 1 h. The permeabilized cells were immunoblotted with rabbit anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody to detect Nedd4-2. Cultured neonatal rat cardiomyocytes were transfected with GFP-tagged Rab4. Forty-eight hours after transfection, cells were fixed and permeabilized. ERG (rat I Kr protein) was labeled with anti-hERG primary antibody (C-20) and Alexa Fluor 594-conjugated secondary antibody. Nedd4-2 was labeled with anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated secondary antibody in separate sets of cells. Images were acquired using a Leica TCS SP2 Multi Photon confocal microscope (Leica, Germany).
All data are expressed as the mean Ϯ S.E. One-way analysis of variance followed by Newman-Keuls post hoc tests (GraphPad Prism), and two-tailed paired or unpaired Student's t test were used to determine the statistical significance between control and experimental groups. A p value Յ0.05 was considered significant. Fig. 1 demonstrates the effect of overexpression of various Rab GTPases on the expression level of hERG channels. The hERG proteins extracted from hERG-HEK cells displayed two bands with molecular masses of 155 and 135 kDa, representing the mature, fully glycosylated form on the plasma membrane and the immature, core-glycosylated form in the endoplasmic reticulum, respectively (5,8). Among the Rab GTPases examined (Rab1, Rab4, Rab5, Rab7, and Rab11), only Rab4 significantly reduced the expression level of the 155-kDa hERG protein. This result is surprising because if Rab4 mediates rapid hERG recycling, an increase in the 155-kDa hERG expression level is expected. As shown in Fig. 1A, overexpression of Rab4 significantly reduced the intensity of the 155-kDa hERG band without affecting the 135-kDa hERG band. Overexpression of Rab4 did not affect the expression level of Kv1.5 or EAG channels stably expressed in HEK 293 cells (Fig. 1C). Consistent with the data obtained using Western blot analysis, Rab4 decreased I hERG but not I Kv1.5 or I EAG (Fig. 1,  B and D). Thus, among the potassium channels Kv1.5, EAG, and hERG, Rab4 selectively targets the hERG channel. Although Rab4 decreased the hERG current amplitude, it did not affect the biophysical properties of I hERG . The half-activation voltage and slope factor of I hERG were Ϫ3.1 Ϯ 0.3 mV and 7.1 Ϯ 0.3 mV in control cells, and Ϫ6.5 Ϯ 0.9 mV and 7.9 Ϯ 0.7 mV in Rab4-transfected cells (n ϭ 4 -8 cells, p Ͼ 0.05).

Overexpression of Rab4 Increases the Ubiquitination of hERG Protein and Enhances the Interaction between hERG and Ub
Ligase Nedd4-2-Our data in Fig. 1 show that overexpression of Rab4 leads to a decrease in mature hERG channel levels. Rab4 is natively expressed in HEK cells. To confirm the role of Rab4 in hERG expression, we knocked down endogenous Rab4 in hERG-HEK cells. As shown in Fig. 2A, knockdown of Rab4 led to a significant increase in mature hERG expression.
Ubiquitination is a process involving the covalent binding of Ub to its target proteins. The best characterized consequence of ubiquitination is the triggering of internalization and/or degradation of the target proteins (27,28). To determine the involvement of ubiquitination in the Rab4-mediated reduction in the 155-kDa hERG expression, we examined the effects of Rab4 overexpression on the Ub-hERG interaction using co-immunoprecipitation analysis. hERG-HEK cells were transfected with pcDNA3 (control) or Rab4 plasmid. To inhibit protein degradation, the proteasome inhibitor ALLN (50 M) was added to the culture medium. Twenty-four hours after transfection, whole-cell proteins were extracted. An anti-Ub antibody was used to immunoprecipitate Ub and its associated proteins. The precipitated proteins were immunoblotted to detect for hERG expression. As shown in Fig. 2B, Rab4 overexpression significantly enhanced the Ub-hERG interaction as evidenced by the more intense ubiquitinated hERG band in Rab4-transfected cells than that in control cells. A hERG band in anti-Ub antibody-precipitated proteins slightly higher than 155 kDa is present, which may reflect monoubiquitinated mature hERG channels. The nature of other enhanced bands with molecular masses smaller than 135 kDa in Rab4-transfected cells is unknown and may reflect fragmented hERG proteins during degradation. Our previous work has also shown that Ub ligase Nedd4-2 only targets the mature (155-kDa) hERG on the plasma membrane for degradation (13).
Transferring ubiquitin to its target proteins, known as ubiquitination, involves a series of enzymes including Ub ligases such as Nedd4-2 that recognizes and labels target proteins with Ub (29). Nedd4-2 plays a critical role in regulating several plasma membrane proteins including epithelial sodium channel ENaC, cardiac voltage-gated sodium channel Nav1.5, potassium channel KCNQ1, and neuronal voltage-gated sodium channel (Na v ) (30 -34). Particularly, we and others have shown that Nedd4-2 interacts with hERG to degrade the mature form   7). To confirm that Rab4 enhanced Ub-hERG interaction, GAPDH was used as a control for the anti-Ub antibody (left panel) in the immunoprecipitation assay. As well, HEK cells were used as control for hERG-HEK cells (right panel). C, Rab4 enhances the hERG-Nedd4-2 interaction. hERG-HEK cells were transfected with pcDNA3 (Ctrl) or Rab4, then cultured for 24 h. Whole-cell lysates were immunoprecipitated with anti-hERG antibody (N-20), and the precipitates were immunoblotted with anti-Nedd4-2 antibody (n ϭ 3).
Disrupting the hERG-Nedd4-2 Interaction Eliminates the Effect of Rab4 on hERG Channels-To determine whether Rab4 regulates hERG expression via Nedd4-2, we investigated the effects of Rab4 overexpression on mutant hERG channels whose Nedd4-2 binding motif is disrupted. As shown in Fig. 3A, both the point mutation Y1078A (PY motif disrupted) and the C-terminal truncation mutation ⌬1073 (PY motif removed) were able to essentially remove the effects of Nedd4-2 on the expression of hERG channels. Also, we have previously shown that Nedd4-2 overexpression decreases I hERG , and both Y1078A and ⌬1073 mutations eliminate the Nedd4-2-induced I hERG reduction (13). Disrupting the Nedd4-2 binding motif also abolished the effects of Rab4 overexpression on hERG channels (Fig. 3B). Consistent with the Western blot analyses, electrophysiology experiments revealed that whereas Rab4 overexpression decreased WT I hERG by 50% (from 1.39 Ϯ 0.14 nA, n ϭ 18, to 0.70 Ϯ 0.09 nA, n ϭ 14; p Ͻ 0.01), it did not significantly affect ⌬1073 I hERG (0.94 Ϯ 0.13 nA, n ϭ 14, in control, versus 0.92 Ϯ 0.16 nA, n ϭ 9, in Rab4-transfected cells; p Ͼ 0.05). Thus, the Rab4-mediated decrease in hERG expression in the plasma membrane requires Nedd4-2.
Overexpression of Rab4 Increases Nedd4-2 Expression-To investigate the mechanisms of Nedd4-2 involvement in the Rab4-mediated reduction in hERG expression, we examined the effect of Rab4 on the expression of Nedd4-2. As shown in Fig. 4, overexpression of Rab4 significantly enhanced the expression level of Nedd4-2 as revealed by Western blotting (Fig. 4A) and immunocytochemical analysis (Fig. 4B). To further confirm the effects of Rab4 on Nedd4-2 expression, we also knocked down Rab4 using siRNA transfection. Knockdown of Rab4 in HEK cells resulted in a significant decrease in the Nedd4-2 expression level (Fig. 4C). In our blots with the Nedd4-2 antibody from Cell Signaling, endogenous Nedd4-2 extracted from HEK cells displayed two bands with molecular masses of 110 and 120 kDa (Figs. 3A, 4, A and C, and 5, A and B). Although the nature of these two bands warrants further investigation and may represent a mixture of Nedd4-2 and its modified forms (ubiquitinated or apo-Nedd4-2), two reasons prompted us to focus on the 110-kDa form of Nedd4-2. First, as shown in Fig. 2C, when the interaction between hERG and Nedd4-2 was examined using co-immunoprecipitation analysis in hERG-HEK cells, only the 110-kDa band was detected in proteins precipitated with an anti-hERG antibody. Second, as shown in Fig. 3A, transfection of the Nedd4-2 plasmid into hERG-HEK cells led to Nedd4-2 overexpression which displayed a molecular mass of 110 kDa.
Rab4 Increases Nedd4-2 Expression by Inhibiting Nedd4-2 Degradation-Nedd4-2 binds to the PY motif of target proteins to mediate protein ubiquitination/degradation (30). Interestingly, Nedd4-2 itself also possesses a PY motif within its catalytic HECT (homologous to E6-associated protein C terminus) domain. When Nedd4-2 is not interacting with its substrates, the PY motif of Nedd4-2 binds weakly to the WW domain of the same molecule, leading to the autoinhibitory state of Nedd4-2. However, after targeting its substrates, Nedd4-2 exposes its PY motif to the WW domain of other Nedd4-2 proteins, leading to self-ubiquitination and degradation (35,36). To test the possibility that Rab4 increases Nedd4-2 by interfering with Nedd4-2 degradation, we treated cells with proteasome inhibitors. We have previously shown that proteasome inhibition can impede Ub-mediated degradation of hERG channels (10). Treatment of hERG-HEK cells with 50 M ALLN (or 10 M MG132, data not shown) increased the level of Nedd4-2 expression, likely by preventing proteasomal degradation. On this elevated back-

Rab4 Regulates hERG via Nedd4-2 FIGURE 4. Rab4 increases the expression level of endogenous Nedd4-2.
A, Rab4 or inactive Rab4N121I affects the expression level of Nedd4-2. The hERG-HEK cells were transfected with pcDNA3, GFP-tagged Rab4, or GFP-tagged Rab4N121I. Twenty-four hours after transfection, whole-cell lysates were extracted and analyzed using Western blotting (n ϭ 4). B, confocal images show that overexpression of Rab4 but not inactive Rab4N121I enhances the expression of endogenous Nedd4-2. hERG-HEK cells were transfected with GFP, GFP-tagged Rab4, or GFP-tagged Rab4N121I (green). Nedd4-2 was labeled with anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated (red) secondary antibody. C, knockdown of Rab4 decreases the expression of endogenous Nedd4-2. The hERG-HEK cells were transfected with scrambled control siRNA or Rab4A siRNA. Twenty-four hours after transfection, whole-cell lysates were extracted and analyzed using Western blotting (n ϭ 4). For analyses in A and C, the intensities of the 110-kDa band of Nedd4-2 from cells in experimental groups were normalized to their respective controls and plotted as relative values. *, p Ͻ 0.05; **, p Ͻ 0.01 versus control. Error bars, S.E. ground, Rab4 overexpression no longer significantly increased Nedd4-2 expression levels (Fig. 5A).
To investigate the effects of Rab4 on the degradation rate of Nedd4-2 directly, we blocked protein synthesis using CHX (10 g/ml) and monitored the Nedd4-2 degradation in control as well as in Rab4-transfected cells. As shown in Fig. 5B, Rab4 overexpression significantly decreased the Nedd4-2 decay rate; after a 12-h treatment with CHX, whereas the Nedd4-2 expression decreased by Ͼ50% in control cells, it only decayed by 15% in Rab4-transfected cells.
Catalytic activity of Nedd4-2 is reported to be required for its self-ubiquitination (35). To confirm this, we examined the Nedd4-2 ubiquitination and the role of catalytic activity in Nedd4-2 degradation by comparing the expression levels of ubiquitinated Nedd4-2 between cells expressing wild-type (WT) Nedd4-2 and those expressing a catalytically inactive Nedd4-2 mutant, Nedd4-2-C801S. It has been shown that the catalytically inactive Nedd4-2 neither catalyzes its substrates nor undergoes self-ubiquitination (35). As shown in Fig. 6A, WT Nedd4-2 but not Nedd4-2-C801S experienced significant ubiquitination. In particular, besides the smeared background which may indicate polyubiquitination, a single band of Ubprecipitated Nedd4-2 which is slightly higher than the Nedd4-2 band in Western blot analysis is present, which should reflect the monoubiquitinated Nedd4-2. Ubiquitination appears to be a prerequisite for the Rab4-mediated increase in Nedd4-2 expression. As shown in Fig. 6B, whereas Rab4 overexpression significantly increased the expression level of Nedd4-2, it failed to increase the expression level of Nedd4-2-C801S. These data raised the possibility that Rab4 interacts preferentially with ubiquitinated Nedd4-2, interferes with its degradation, and promotes its recycling, resulting in an increased expression level of Nedd4-2. Our data shown in Fig. 6C directly support this notion; between Nedd4-2 and Nedd4-2-C801S, Rab4 preferentially co-precipitated with Nedd4-2. Furthermore, the Rab4-precipitated Nedd4-2 band is slightly higher than the Nedd4-2 band in Western blot analysis, suggesting that Rab4 interacts with monoubiquitinated Nedd4-2. Finally, overexpression of Rab4 decreased the ubiquitinated Nedd4-2 (Fig.  6D), which may underlie the slowed degradation, and enhanced expression of Nedd4-2.
The Effect of Rab4 on the Expression and Function of I Kr in Neonatal Rat Cardiomyocytes-Rab4 is natively expressed in cardiomyocytes and serves important functions. By transgenic expression of dominant negative Rab4 S27N in mice, Odley et al. showed that Rab4 mediates recycling of internalized ␤-adrenergic receptors, which is necessary for normal cardiac catecholamine responsiveness and resensitization after agonist exposure (37). Rab4 expression levels have been shown to be altered in certain pathological conditions. Transgenic overexpression of ␤ 2 -adrenergic receptors in mouse hearts leads to heart failure with augmented Rab4 expression levels (38). Increased Rab4 expression is also observed in Akt2 deficiencyinduced cardiomyopathy which is similar to type 2 diabetic cardiomyopathy (39). Transgenic overexpression of Rab4 in the mouse myocardium induces concentric cardiac hypertrophy (40). To determine whether Rab4 expression affects I Kr in cardiomyocytes, we altered Rab4 expression levels by overexpressing Rab4 as well as knocking down Rab4 in neonatal rat cardiomyocytes. As shown in Fig. 7A-C, overexpression of Rab4 decreased the expression level of the mature form of ERG (I Kr protein in rats) whereas knockdown of Rab4 increased the expression level. As well, overexpression of Rab4 significantly decreased the I Kr recorded by whole-cell patch clamp using Cs ϩ permeation to isolate I Kr from other K ϩ currents (Fig. 7D). To confirm the involvement of Nedd4-2 in Rab4-mediated changes in ERG, immunofluorescence microscopy was used to examine the effects of Rab4 overexpression on ERG and Nedd4-2 levels. As shown in Fig. 7E, overexpression of Rab4-GFP led to a decrease in ERG expression and an increase in Nedd4-2 expression.  3). B, effects of Rab4 on the expression levels of Nedd4-2 and catalytically inactive Nedd4-2-C801S (Nedd4-2CS). HEK 293 cells transfected with either Nedd4-2 or Nedd4-2-C801S were co-transfected with pcDNA3 (Ctrl) or Rab4. Western blot analyses were performed 24 h after transfection. The intensities of Nedd4-2 in Rab4-transfected cells were normalized to their respective controls and expressed as relative values (n ϭ 3). *, p Ͻ 0.05 versus control. Error bars, S.E. C, Rab4 interacting with Nedd4-2 but not Nedd4-2-C801S (Nedd4-2CS). Whole-cell lysates from HEK 293 cells transfected with Nedd4-2 or Nedd4-2-C801S were immunoprecipitated with anti-Rab4 antibody. The precipitates were immunoblotted with anti-Nedd4-2 antibody (n ϭ 4). D, effects of Rab4 on Ub-Nedd4-2 interactions. Whole-cell proteins from HEK 293 cells co-transfected with Nedd4-2 plus pcDNA3 (Ctrl) or Nedd4-2 plus Rab4 were immunoprecipitated with anti-Ub antibody. The precipitates were immunoblotted with anti-Nedd4-2 antibody (n ϭ 6). Rab4 overexpression significantly decreased the ubiquitinated Nedd4-2. In A, C, and D, a fraction of protein from Nedd4-2-transfected cells was immunoblotted with anti-Nedd4-2 antibody to show Nedd4-2 expression as a positive control.

DISCUSSION
The hERG potassium channel plays a critical role in the repolarization of the cardiac action potential. The whole-cell current of any type of ion channel is determined by single channel activities (gating, i.e. open versus closed) and the total number of functional channels at the plasma membrane. Studies have shown that mutations in hERG or drugs can decrease the hERG density at the plasma membrane, leading to a diminished I hERG , which causes long QT syndrome (7,41). However, it is unknown how the density of hERG channels on the plasma membrane is regulated. We previously demonstrated that hERG channels undergo endocytic degradation which is accelerated under hypokalemic conditions (5). Because Rab4 has been shown to mediate the rapid recycling of various endocytosed membrane proteins (20,21), we investigated the effects of various Rab GTPases, especially Rab4, on hERG expression. Our data show that Rab4 selectively decreases the plasma membrane-localized 155-kDa hERG proteins (Figs. 1A and 3B). We demonstrated a novel regulatory mechanism where Rab4 decreases the expression of hERG potassium channels through enhancing the expression level of the Ub ligase Nedd4-2, which mediates hERG ubiquitination and degradation.
Covalent attachment of single or multiple Ub molecules to a target protein is known as ubiquitination. The WW domain of Nedd4-2 binds to the PY motif of the target protein to mediate protein ubiquitination. Interestingly, although both immature (135-kDa) and mature (155-kDa) hERG channels possess the PY motif, our present as well as previous studies demonstrate that Nedd4-2 selectively targets and induces the degradation of the mature (155-kDa) hERG channels located at the plasma membrane (13). Similar results were also published by Albesa et al. (12). The mechanisms for Nedd4-2 to selectively reduce the 155-kDa form of hERG remain to be fully understood, and the following two mechanisms may be involved. First, membrane adaptor proteins such as NDFIP2 (Nedd4 family-interacting protein 2) function as both a recruiter and a strong activator of the Nedd4 family (42). We have also shown previously that caveolin-3 recruits Nedd4-2 to the plasma membrane to interact with mature hERG channels (13). Second, Nedd4-2 possesses a C2 domain in its N terminus, which binds to the cellular membrane in a Ca 2ϩ -dependent manner (43). The C2 domain of Nedd4-2 binds to its HECT domain in the inactive state. Calcium-dependent binding of the C2 domain to the membrane phospholipids dissociates the C2 domain from the HECT domain, leading to the activation of Nedd4-2 (36). Thus, C2 domain-mediated translocation of Nedd4-2 to the cellular membrane could be responsible for the selective targeting of Nedd4-2 to the plasma membrane-localized mature hERG channels.
Nedd4-2 also possesses a PY motif in its catalytic HECT domain (35). In the absence of other substrates, the PY motif (LPXY) of Nedd4-2 binds weakly to its own WW domains FIGURE 7. Effects of Rab4 on the function and expression of I Kr in neonatal rat cardiomyocytes. A and B, overexpression of Rab4 decreases whereas knockdown of Rab4 increases the expression of ERG in neonatal rat cardiomyocytes. Cultured neonatal rat cardiomyocytes were transfected with pcDNA3 or Rab4-GFP; control siRNA or Rab4A siRNA. Forty-eight hours after transfection, whole-cell lysates were extracted and analyzed using Western blotting. Wholecell proteins from hERG-HEK cells were also loaded in the Western blots to show hERG expression. C, summarized relative intensities (Intensity-Rel) of the mature ERG band are compared with their respective controls (n ϭ 4 -6; *, p Ͻ 0.05). Error bars, S.E. D, overexpression of Rab4 decreases I Kr . Cultured neonatal rat cardiomyocytes were transfected with GFP or GFP-tagged Rab4 (Rab4-GFP). Cells expressing GFP were selected for recording Cs ϩ -mediated I Kr (I Kr-Cs ). The summarized amplitudes of the tail currents of I Kr-Cs were plotted (n ϭ 21 in control and 22 in Rab4-transfected cells). *, p Ͻ 0.05 versus control. E, confocal image shows that overexpression of Rab4 decreases the native ERG expression and increases the Nedd4-2 expression. Cultured neonatal rat cardiomyocytes were transfected with GFP-tagged Rab4 (green). ERG was labeled with anti-hERG (C-20) primary antibody and Alexa Fluor 594-conjugated (red) secondary antibody. Nedd4-2 was labeled with anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated (red) secondary antibody. Detection of ERG and Nedd4-2 was performed independently.
We propose that the ubiquitination of Nedd4-2 takes place at the plasma membrane when it interacts with and mediates ubiquitination of hERG channels. Ubiquitinated Nedd4-2 can be either degraded along the degradation pathways or recycled back to the plasma membrane (Fig. 8). Rab4 is known to be associated with early endosomal recycling and the transport of internalized proteins back to the plasma membrane (37,44). Thus, Rab4 may facilitate the recycling of Nedd4-2, leading to decreased degradation and increased expression. The increased Nedd4-2 consequently leads to an enhanced degradation of mature hERG channels (Fig. 8). Our data show that disrupting the interaction between Nedd4-2 and hERG completely abolishes the effects of Rab4 on hERG channels, indicating that Nedd4-2-mediated regulation is the primary pathway for Rab4 to regulate hERG, and the direct effects of Rab4 on hERG trafficking seem to be negligible.
Our findings may have broad implications for the Nedd4-2 target proteins which possess the PY motif. For example, pre-FIGURE 8. Proposed scheme illustrating that Rab4 increases Nedd4-2 expression by facilitating Nedd4-2 recycling. Prior to targeting its substrates, Nedd4-2 remains inactive with its WW domain binding to the PY motif within the catalytic HECT domain of the same molecule. The WW domain of Nedd4-2 binds to the PY motif of hERG channels in the plasma membrane, resulting in Nedd4-2 activation, which mediates hERG ubiquitination and endocytic degradation. Meanwhile, activation of Nedd4-2 leads to the expose of its own PY motif within the HECT domain to other Nedd4-2 molecules, resulting in the ubiquitination of Nedd4-2. The ubiquitinated Nedd4-2 can be either degraded along the degradation pathways or recycled back to the plasma membrane. Rab4 facilitates the recycling and consequently reduces degradation of Nedd4-2, resulting in an increased Nedd4-2 level. The increased Nedd4-2 level causes a decreased expression of mature hERG channels at the plasma membrane.
vious studies have demonstrated that Rab4 overexpression decreases the plasma membrane expression of epithelial sodium channel (45) and cystic fibrosis transmembrane conductance regulator (46). However, the mechanisms for Rab4 in these regulations are not known (45,46). Because both epithelial sodium channel and cystic fibrosis transmembrane conductance regulator are Nedd4-2 substrates (30,47), it is likely that Rab4 also regulates these two channels via Nedd4-2. In addition, cardiac and neuronal Na ϩ channels, cardiac K ϩ channel KCNQ1, and neuronal K ϩ channels KCNQ2/3/5 are among the substrates of Nedd4-2 (32)(33)(34)48). Besides the direct effects on these channels, Rab4 is expected to regulate these channels via altered Nedd4-2 expression levels. Finally, Nedd4-2 is involved in a variety of cellular processes including neuronal development and cell growth (49,50). Thus, the Rab4-mediated regulation of Nedd4-2 may have a wide impact on various cellular processes.
Rab4 expression is highly variable and regulated in cardiomyocytes (37)(38)(39). Our data demonstrate that Rab4-mediated alterations in ERG expression also exist in cardiomyocytes (Fig. 7). A previous study has demonstrated that cardiac Rab4 is up-regulated in a dilated cardiomyopathy model overexpressing ␤ 2 -adrenergic receptors (38). Up-regulation of Rab4 expression is also observed in ventricular tissues of Akt2knock-out mice which develop a syndrome similar to diabetes mellitus type 2 cardiomyopathy (39). Transgenic overexpression of Rab4 in the mouse myocardium induces concentric cardiac hypertrophy (40). Cardiomyopathy as well as heart failure are closely associated with QT prolongation and ventricular arrhythmias (51)(52)(53)(54)(55). Specifically, a link between depressed hERG channel function and abnormal QT prolongation in diabetic rabbits was also reported (56). Thus, our study raised the possibility that elevated Rab4-mediated reduction in hERG expression may play a role in the development of QT prolongation in patients with cardiomyopathy and heart failure.
In summary, the present study has revealed a novel mechanism for Nedd4-2 and hERG regulation; Rab4 increases Nedd4-2 expression, which consequently decreases the expression of mature hERG channels. The Rab4-mediated Nedd4-2 regulation could impact other cellular processes that are regulated by Nedd4-2.