Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.

PKC and PKA 18 , decreased the expression and activity of Kv1.3 in activated macrophages and CY15 dendritic cells. Although PKC mostly triggers internalization of channels and transporters [19][20][21][22] , ADO stabilizes KATP channels at the membrane surface in a PKC-dependent manner 23 . With this debate in mind, we investigated whether the modulation of Kv1.3 was consequence of a specific PKC-mediated endocytosis. HEK-293 cells, similarly to macrophages [24][25][26] , endogenously express the A 2B subtype of adenosine receptors 27 . Therefore, this cell line is a good model for dissecting the ADO-dependent PKC signaling.
We further analyzed the participation of a PKC-dependent mechanism by using the PKC agonist phorbol 12-myristate 13-acetate (PMA). The presence of the PKC inhibitor bisindolylmaleimide (BIM) hampered the internalization of channels ( Fig. 2A-D,G), indicating that ADO triggered Kv1.3 endocytosis via stimulation of PKC. Similarly, specific PMA-dependent PKC activation also promoted the redistribution of Kv1.3 from the cell surface to vesicular structures ( Fig. 2E-G). BIM abolished this effect, indicating that, similar to ADO-induced endocytosis, PMA-associated endocytosis was dependent on PKC ( Fig. 2B,D,F,G). This mechanism was indeed concomitant to an activation of the PKC. We analyzed the phosphorylation of PKCε because this isoform participates in TNF-α -dependent pro-inflammatory responses in HEK cells 28 and during the LPS-induced and MCSF-dependent activation in macrophages [29][30][31] . ADO (Fig. 2H) and PMA (Fig. 2I) augmented the phosphorylation of PKCε (~2 fold increase), without changes in total PKC. Again BIM effectively hampered (~50% decrease) PKC phosphorylation in both cases. Next, we monitored the time-course of Kv1.3 distribution under persistent PKC activation. PMA steadily induced the endocytosis of Kv1.3 (p < 0.0001, One-way ANOVA), which was almost completely internalized within 30 minutes (Fig. 2J-N). Supporting HEK 293 cells, similar mechanisms functioned in mononuclear phagocytes (see Fig. 1), because a 30 min incubation with ADO ( Fig. 3O-Q) vesicularized Kv1.3 in CY15 dendritic cells.
We next analyzed whether the Kv1.3 internalization was associated with changes in Kv currents in Kv1.3-YFP stable HEK-293 cells. Depolarizing pulses elicited K + currents, and 30 min incubation with PMA reduced those currents by 49%. BIM counteracted this effect, demonstrating a specific dependence on the PKC stimulation ( Fig. 3A). The I-V plots showed that PKC activation efficiently reduced current densities at all activation voltages ( Fig. 3B) (p < 0.001, two-way ANOVA). We further analyzed the specific Kv1.3 C-type inactivation (Fig. 3C-F). This hallmark, which is the result of the cooperative interaction of all subunits within the complex, remained unaltered 32 . Thus, the τ of current decay (in ms) was similar being 720 ± 14, 698 ± 14, 742 ± 30 and 741 ± 11 for Control (Fig. 3C), PMA (Fig. 3D), Control + BIM (Fig. 3E) and PMA + BIM (Fig. 3F) respectively. Taken together, these results further supported the down-regulation of Kv1.3, mostly governed by a PKC-mediated decrease of channel subunits at the cell surface, rather than major changes in Kv1.3 biophysics upon PKC activation.
PKC-dependent Kv1.3 endocytosis is a clathrin-mediated mechanism. We next dissected the mechanisms of PKC-dependent Kv1.3 endocytosis (Fig. 4). After 5 minutes of PMA incubation, Kv1.3 colocalized with AP2 (Adaptor-related Protein complex 2) and Clathrin, components of the clathrin-coated pit (CCP) . β -Actin was used as a control reference. Values are mean ± SE of 4-5 independent experiments. Statistical analysis by One-Way ANOVA (P < 0.001) with a Tuckey post-test (*, p < 0.05; ***, p < 0.001). (E) CY15 cells were held at − 80 mV, and voltage-dependent K + currents were elicited by a 250 ms depolarizing pulse from − 80 mV to + 60 mV. Black traces, CY15 cells in the absence of ADO; grey traces, cells in the presence of ADO. (F) Current density vs. voltage for outward K + currents in CY15 cells. Currents were elicited by 250 ms pulses from − 60 mV to + 80 mV in 10 mV steps. Circles, control cells in the absence (○ ) or the presence (• ) of ADO. Squares, LPS-treated cells in the absence (□ ) or the presence (■ ) of ADO. Statistical analysis was performed by Two-Way ANOVA (p < 0.001, LPS vs control, CTR + ADO and LPS + ADO) with a Bonferroni post-test (p < 0.05, LPS vs all other groups at − 10 mV; p < 0.01, LPS vs all other groups at 0 mV; p < 0.001, LPS vs all other groups from 10 to 80 mV). Values are shown as the mean ± SE (n = 5-10 independent cells).   (Fig. 4Q). Clathrin and Dyn II also participate in the exocytic pathway of membrane proteins 33 ; therefore, some perinuclear Kv1.3 accumulation was observed (arrowheads in Fig. 4L,M,O,P). Altogether, this evidence supports that CCP-mediated endocytosis was the main pathway for PKC-dependent Kv1.3 internalization.
PSD-95 association stabilizes Kv1.3 at membrane raft microdomains and prevents PKC-mediated channel endocytosis. PKC and Kv1.3 are recruited into the IS, fine-tuning the T-cell activation 10,11 . Moreover, the IS concentrates lipid raft microdomains as signaling platforms 10 . Kv1.3 interacts with proteins from the MAGUK family such as PSD-95, which recruits the channel into the IS 37 . Impaired IS

PKC-dependent Kv1.3 endocytosis targeted the channel for lysosomal degradation.
Our results in macrophages and CY15 dendritic cells suggested that the abundance of Kv1.3 was decreased under ADO-dependent PKC stimulation (Fig. 1). Therefore, to study in more detail whether Kv1.3 clathrin-mediated internalization ends in a lysosomal-associated degradative intracellular compartment, the PKC-dependent Kv1.3 endocytic mechanisms were further analyzed. After 15 minutes, Kv1.3 colocalized with Transferrin Receptor (Trsf-R), which is used as a CCP marker (Fig. 7Aa-Bd). After 30 minutes of PMA incubation, Kv1.3 was distributed within early endosomes, identified by Early Endosomal Antigen 1 (EEA1) (Fig. 7Ca-Dd). Finally, 120 minutes of PKC activation targeted Kv1.3 to lysosomes stained with LysoTracker ® Red (Fig. 7Ea-Fd). Concomitantly, 4 hours of PMA incubation in the presence of 100 μ g/ml CHX triggered a massive decrease of Kv1.3 expression (Fig. 7Ga-Gc). Further, when also inhibiting lysosomal degradation using the vacuolar-type H + -ATPase inhibitor BafA1 (20 μ M), the rapid PKC-associated decrease in Kv1.3 abundance was halted (Fig. 7H). Overall, our results suggest that PKC activation, either by ADO or by PMA, triggered a down-regulation of cell-surface Kv1.3 via CCP that targets the channel for lysosomal degradation.

Discussion
Adenosine (ADO), which activates PKC signaling pathways, is an endogenous regulator in many physiological processes 7,8,43,44 . PKC modulates Kv1.3 activity, which is essential for a proper immune response 14,45 3,46 , elucidating the mechanism by which ADO-dependent PKC-activation modulates this channel is essential for understanding the immune response. Therefore, we analyzed the PKC-based mechanisms that control the cell-surface levels of Kv1.3 as an efficient way to regulate the function of this ion channel. ADO, activating PKC, initiates a signaling cascade that modulates the cell-surface abundance of Kv1.3 by channel endocytosis. In addition, PSD-95 stabilizes Kv1.3 into lipid raft microdomains and protects the channel against internalization. Kv1.3 endocytosis is a CCP-dependent mechanism that drives the channel to lysosome-associated degradative intracellular compartments and requires previous ubiquitination by the E3 ubiquitin ligase Nedd4-2.
While activation of mononuclear phagocytes increases Kv1.3, anti-inflammatory agents decrease channel expression 14,16,47 . ADO, via A 2A and A 2B receptors, is a potent endogenous immunosuppressor 7,15 , and it decreases Kv1.3 levels in macrophages and dendritic cells. In addition, ADO induced internalization of the channel in  In contrast, an ADO-mediated PKC-dependent mechanism stabilizes Kir6.2 channels at the myocyte plasma membrane 23 . However, PKC activation induces internalization of the epithelial sodium channel (ENaC) and the dopamine transporter (DAT) by the same clathrin-mediated endocytosis (CME) mechanism as Kv1.3 [48][49][50] . CME also participates in the Kv1.2 and Kir1.1 endocytic pathways in neurons and renal cells 51,52 . Our results support that the down-regulation of Kv1.3 would trigger immunosuppression via PKC-dependent CME in leukocytes. This mechanism accelerates the degradation of the channel by targeting it to the lysosomal compartment after PKC-dependent stimulation. We further demonstrated that this process requires direct ubiquitination of Kv1.3 by the E3 ubiquitin ligase Nedd4-2. Recent evidence has shown that Nedd4-2 also drives Kv1.3 toward proteasomal degradation 41 . Thus, efficient Nedd4-2-dependent ubiquitination triggers the down-regulation of Kv1.3 via lysosomes and proteasomes, likely acting as redundant and complementary pathways. PKC activation triggers endocytosis and ubiquitination of proteins such as DAT, the glutamate transporter GLT1, the cationic amino acid transporter CAT-1 and aquaporin-2 (AQP2) 22,50,53,54 . Nedd4-2 downregulates Kv1.3 currents, but no direct ubiquitination of the channel had been demonstrated until now 41,42 . The absence of a canonical PY motif together with the irrelevance of a SH3 signature and several lysines within the C-terminal domain of Kv1.3 would suggest alternative mechanisms of association 41 .
In T-cells and macrophages, Kv1.3 is targeted to lipid raft membrane microdomains, where it is concentrated upon cellular activation 11,55 . Altered raft-associated Kv1.3 localization in the IS occurs at the onset of the disease lupus erythematosus 38 and a minor raft targeting of Kv1.3 modifies the physiological response 34,47 . We demonstrated that PKC activation displaced Kv1.3 from lipid rafts, similar to what was described for the transporter NET 56 . However, unlike that of NET, the PKC-dependent endocytosis of Kv1.3 is independent of caveolae/lipid raft internalization. Several ion channels, such as TRPV5 and Kir6.1, use the caveolae-dependent internalization machinery 35,57 . TRPV5 endocytosis is inhibited by caveolin-1 knockdown 57 . However, similar to the cationic amino acid and dopamine transporters (CAT1 and DAT, respectively), Kv1.3 was internalized via CCP and independently of caveolae/rafts 22,50 . In this context, the localization of Kv1.3 in and out of rafts would have important physiological consequences. Therefore, recruitment and/or stabilization of the channel at the proper location by accessory proteins, such as caveolins and MAGUKs, would influence the immune response. PKC and Kv1.3 are distributed into lipid raft membrane microdomains within T-lymphocytes, and they are recruited to the IS during T-cell activation 11 . Moreover, PKC and Kv1.3 are part of a signalplex that also includes the tyrosine kinase p56lck, adaptor proteins such as Kvβ 2 and hDlg (human homolog of the Drosophila discs large tumor suppressor protein), PSD-95 (postsynaptic density 95), ZIP-1 (Zrt/Irt-like protein) and ZIP-2, and the accessory protein CD4 58 . This cluster facilitates the phosphorylation of Kv1.3, thereby modulating its function. Our results show that PSD-95 association redistributes Kv1.3 into less-mobile membrane microdomains compatible with lipid rafts.
In T-lymphocytes, PSD-95 interacts with Kv1.3, inducing clustering and recruiting Kv1.3 into the IS 37 . Similarly, PSD-95 recruits Kv1.4 into lipid rafts, inhibiting the internalization of Kv1.4 59 . A dynamic partitioning model would explain the behavior of raft proteins, suggesting movements in and out of raft domains in a steady-state equilibrium. This spatial distribution would permit proteins to transiently populate raft domains as well as to undergo diffusion outside of rafts. It is tempting to speculate that Kv1.3 would exit rafts being internalized via CCP during immunosuppression. In fact, Kv1.5, acting as an immunosuppresor, influences Kv1.3 by displacing heteromeric Kv1.3/Kv1.5 channels out of rafts in macrophages 34,47,60 . In this scenario, PKC would enhance the exit of Kv1.3 from raft areas and/or facilitate its distribution into clathrin-accessible regions to be endocytosed. In fact, PMA-induced PKC-activation in T-lymphocytes inhibits Kv1.3-dependent K + currents 6 . Therefore, a functional Kv1.3-PSD-95 interaction is required not only for correct Kv1.3 redistribution in the vicinity of IS formation but also to prevent a massive internalization of the channel as a consequence of PKC-dependent immunosuppressive insults.
New treatments targeting inflammation-mediated organ dysfunction associated with autoimmune diseases are worth investigation. After persistent activation, mononuclear phagocytes play a pivotal role, supporting the characterization of ADO as an immunomodulatory agent. Adenoreceptor stimulation attenuates inflammation-mediated damage by down-regulating phagocytic activity and preventing excessive respiratory burst activation. For example, the inflammatory response is partially responsible for the damage associated with The localization of Kv1.3-YFP into lysosomes was confirmed by LysoTracker (LysoTr) staining. Color code: Kv1.3, green; intracellular compartment marker, red. Yellow in merge panels (Ac, Bc, Cc, Dc, Ec, Fc) indicates colocalization. Insets in panels Bc, Dc and Fc magnify specific regions of interest, indicated by square outlines.  reperfusion of ischemic tissues. ADO modulation has been demonstrated in ischemia/reperfusion injury 61 . In this respect, selective inhibition of A 2B enhances intestinal inflammation and injury following ischemia/reperfusion, whereas specific A 2B agonist treatment protects against intestinal injury 62 . A 2B receptor agonists reduce myocardial ischemia/reperfusion damage by promoting anti-inflammatory M2 macrophages together with decreases in M1 macrophage and neutrophil infiltration in re-perfused hearts 63 . In this context, Kv1.3 is a viable pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke 64 . Our results shed light on the molecular mechanism underlying the anti-inflammatory function of A 2B agonist therapy via the modulation of Kv1.3.

Methods
Expression plasmids and site-directed mutagenesis. The rat Kv1.3 in the pRcCMV construct was provided by T.C. Holmes (University of California, Irvine, CA). The channel was subcloned into pEYFP-C1 (Clontech). The rKv1.3 construct that was externally tagged with HA between S3 and S4 was obtained from D.B. Arnold (University of Southern California, CA). For some experiments, the HA-Kv1.3 channel was further subcloned into the pEYFP-C1 plasmid, generating the double-tagged HA-Kv1.3-YFP channel. All Kv1.3 mutants were generated in the pEYFP-Kv1.3 channel. Single and multiple Kv1.3 mutants were generated using the QuikChange site-directed and multi-site-directed mutagenesis kits (Stratagene). All mutations were verified using automated DNA sequencing. Myc-PSD-95 was a kind gift from Dr. F. Zafra (Centro de Biología Molecular Severo Ochoa, Madrid).

Cell culture, transfections and incubations.
Unless specified, all reagents were purchased from Samples were incubated overnight with the specified antibody (4 ng/μ g protein) at 4 °C with gentle agitation. Next, 30 μ l of protein G-Sepharose 4 fast flow (GE Healthcare) was added to each sample, and the samples were incubated for 4 h at 4 °C. Beads were removed by centrifugation at 1,000 × g for 30 s at 4 °C, washed four times in NHG, and resuspended in 80 μ l of SDS sample buffer.
Lipid raft isolation was performed as previously described 34 . Briefly, samples were homogenized in MES (2-Morpholino ethanesulfonic acid)-buffered saline (24 mM MES, pH 6.5, and 0.15 mM NaCl) plus 1% Triton X-100 and centrifuged at 3,000 g for 5 min at 4 °C. Next, sucrose was added to achieve a final concentration of 40%. A 5-30% linear sucrose gradient was layered on top and further centrifuged (39,000 rpm) for 20-22 h at 4 °C in a Beckman SW41 Ti swinging bucket rotor. Gradient fractions (1 ml) were collected from the top and analyzed by Western blot.
Confocal microscopy and subcellular compartment identification. Cells fixed with 4% paraformaldehyde in PBS for 10 min were further permeabilized using 0.1% Triton X-100 for 10 min. After a 60 min incubation with a blocking solution (PBS, 10% goat serum, 5% non-fat dry milk), cells were incubated for 60 min with anti-Clathrin (clathrin heavy chain, 1/100, BD Bioscience), anti-α subunit of AP2 (Adaptor-related Protein complex 2, 1/500, AP.6, American Type Culture Collection), anti-Transferrin receptor (1/1,000, Abcam) or anti-EEA1 (Early Endosomal Antigen 1, 1/1,000, BD Bioscience) in PBS, 10% goat serum and 0.05% Triton. For the extracellular distribution of HA-Kv1.3-YFP, cells were incubated with anti-HA (1/1,000, Sigma) under non-permeabilizing conditions. Next, cells were further incubated for 45 min with an Alexa Fluor secondary antibody (1:500, Molecular Probes) in PBS and BSA (2%). All experiments were performed at 21-23 °C (RT). In some experiments, cells were washed with PBS at 4 °C and stained with LysoTracker ® red (1/1,000, Molecular Probes) for 30 min at 4 °C. Staining with FITC-labeled cholera toxin β subunit (CTXβ ) for lipid raft microdomains was performed under non-permeabilized conditions. Cells washed with PBS were stained with FITC-CTXβ for 30 min at 4 °C. Subsequently, cells were washed and fixed as above. Cells were examined with a 63x oil immersion objective on a Leica TCS SL laser-scanning confocal microscope. All offline image analyses were performed using a Leica confocal microscope, Image J software and Sigma Plot. The level of endocytosis was quantified analyzing the number of intracellular vesicle accumulation by using the automatic particle counting protocol of the Image J software and setting threshold around 75% to discard the membrane surface mask. siRNA transfections. Synthetic siRNAs for Clathrin, dynamin II and the missense negative control (Mock) were purchased from Dharmacon. Duplexes were resuspended in 1x siRNA universal buffer (Dharmacon) to 20 μ M. HEK-293 cells expressing the stable Kv1.3-YFP channel were grown in six-well plates to 50% confluence. Cells were transfected with siRNA duplexes to a final concentration of 120 nM in 5 μ l DharmaFECT1 reagent (Dharmacon, Inc). After 36 h, a second transfection was performed, and the cells were replated into 12-well plates on the following day for internalization experiments. The efficiency of knockdown was evaluated by Western blotting. Mock-and siRNA-transfected cells were processed for immunofluorescence as described above.
Antibody-feeding endocytosis assay. Cells grown on glass coverslips were incubated with 1-2 μ g/ml of anti-HA11 (1/1,000, Covance) in DMEM for 30-60 min at RT, washed twice and incubated at 37 °C in the presence or in the absence of 1 μ M PMA for 30 min. The cells were then washed with ice-cold Ca 2+ /Mg 2+ -free PBS (CMF-PBS) and fixed with freshly prepared 4% paraformaldehyde for 8 min at room temperature. Cells were stained with secondary anti-mouse antibody conjugated with Cy5 (5 μ g/ml, saturating concentration) in CMF-PBS containing 0.5% BSA at RT for 60 min to occupy surface HA11. After washings, the cells were permeabilized by 10 min of incubation in CMF-PBS containing 0.1% Triton X-100 at RT and then incubated with the same secondary antibody conjugated with Cy3 (1 μ g/ml, non-saturating concentration) for 60 min to stain internalized HA11. Both primary and secondary antibody solutions were precleared by centrifugation at 100,000 × g for 20 min. After staining, cells were washed, and the coverslips were mounted in Mowiol (Calbiochem).
Electrophysiology. Whole-cell currents were recorded using the patch-clamp technique in the whole-cell configuration with a HEKA EPC10 USB amplifier (HEKA Elektronik). PatchMaster software (HEKA) was used for data acquisition. We applied a stimulation frequency of 50 kHz and a filter at 10 kHz. The capacitance and series resistance compensation were optimized. In most experiments, we obtained an 80% compensation of the effective access resistance. Micropipettes were made from borosilicate glass capillaries (Harvard Apparatus) using a P-97 puller (Sutter Instrument) and fire polished. The pipettes had a resistance of 2-4 MΩ. For the stably transfected HEK-293 cells, pipettes were filled with a solution containing the following (in mM): 120 KCl, 1 CaCl 2 , 2 MgCl 2 , 10 HEPES, 10 EGTA, 20 D-glucose (pH 7.3 and 280 mOsm/l). The extracellular solution contained the following (in mM): 120 NaCl, 5.4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES and 25 D-glucose (pH 7.4 and 310 mOsm/l). Cells were clamped at a holding potential of -60 mV. To evoke voltage-gated currents, cells were stimulated with 250 ms square pulses from -60 to + 50 mV in 10 mV steps. To analyze the C-type inactivation of Kv1.3, a 5 s depolarizing pulse of + 60 mV was applied. Electrodes for CY15 cells were filled with a solution containing the following (in mM): 84 K-aspartate, 36 KCl, 10 KH 2 PO 4 , 6 K 2 ATP, 5 HEPES, 5 EGTA, and 3 MgCl 2 , pH 7.2. The extracellular solution contained the following (in mM): 136 NaCl, 4 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 10 D-glucose, pH 7.4. CY15 cells were clamped to a holding potential of − 60 mV. To evoke voltage-gated currents, cells were stimulated with 250 ms square pulses ranging from -60 to + 80 mV in 10 mV steps. The peak amplitude (pA) was normalized using the capacitance values (pF). Data analysis was performed using FitMaster (HEKA) and Sigma Plot 10.0 software (Systat Software). All recordings were performed at RT.

Statistics.
Statistical analysis was performed where indicated by means of One-Way or Two-Way ANOVA with Tukey or Bonferroni post-test respectively, Mann-Whitney U test or Student t test by using GraphPad Prism 5 (Graphpad Software Inc.).