The FASEB Journal article fj.13-242032. Published online January 22, 2014.

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Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels Wiebke Wilkars,* Jessica Wollberg,† Evita Mohr,* Mieri Han,* Dane M. Chetkovich,‡,§ Robert Bähring,† and Roland A. Bender*,1 *Institute of Neuroanatomy and †Institute of Cellular and Integrative Physiology, University of Hamburg Medical Center, Hamburg, Germany; and ‡Davee Department of Neurology and Clinical Neurosciences and §Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ⬃100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85–92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2.Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.—Wilkars, W., Wollberg, J., Mohr, E., Han,

ABSTRACT

Abbreviations: CA1, cornu ammonis 1; Co-IP, co-immunoprecipitation; DAPI, 4;6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle medium; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin A; HCN, hyperpolarization-activated cyclic nucleotide-gated; HECT, homologous to the E6-AP, carboxyl terminus; HEK293, human embryonic kidney 293; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; Nedd, neuronal precursor cell-expressed developmentally downregulated; PBS, phosphate-buffered saline; PFA, paraformaldehyde; TRIP8b, tetratricopeptide repeat-containing Rab8b interacting protein 0892-6638/14/0028-0001 © FASEB

M., Chetkovich, D. M., Bähring, R., Bender, R. A. Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels. FASEB J. 28, 000 – 000 (2014). www.fasebj.org Key Words: ion channel 䡠 trafficking 䡠 TRIP8b 䡠 ubiquitination Hyperpolarization-activated cyclic nucleotidegated (HCN) channels, which mediate the hyperpolarization-activated current (Ih), are important determinants of intrinsic excitability in neurons (1–3). The channels are structurally related to the potassium channel superfamily and assemble as homo- or heteromeric tetramers to which 4 pore-forming subunits (HCN1– 4) can contribute, each conferring individual physiological properties to the resulting channels (2, 4). These properties are further regulated through intracellular metabolites (5– 8), covalent post-translational channel modifications (9 –11), or interaction with auxiliary subunits (12–15), thus enabling the channels to respond to a wide variety of cellular signals and to fine tune neuronal responses. A post-translational modification that increasingly receives attention as a mechanism of ion channel regulation is ubiquitination, a complex process requiring 3 sequential enzymatic steps (E1, E2, and E3), through which ubiquitin, a highly conserved, 76-aa polypeptide, is attached to proteins. It was originally described as a signal that targets cellular proteins to rapid degradation, but was later found also to regulate numerous other processes in the cell, including protein trafficking (16, 17). Specificity of ubiquitination in mammals is provided by hundreds of different E3 ubiquitin–protein ligases that recognize relevant target 1 Correspondence: Institute of Neuroanatomy, University of Hamburg Medical Center, Martinistr. 52, 20246 Hamburg, Germany. E-mail: [email protected] doi: 10.1096/fj.13-242032 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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proteins and promote the conjugation of ubiquitin. There are two major E3 classes—RING (really interesting new gene) and HECT (homologous to the E6-AP, carboxyl terminus)—the latter including the neuronal precursor cell-expressed developmentally downregulated (Nedd) 4-like proteins Nedd4-1 and Nedd-4-2, which interact with target proteins through a specific motif, PY (L/PPxY), that is frequently found in the C terminus of voltage-gated ion channels. Both Nedd4-1 and Nedd-4-2 have been shown to interact with and regulate ubiquitination in certain subtypes of ion channels via this motif (18, 19). Here, we provide evidence that Nedd-mediated ubiquitination also plays a role in the regulation of HCN channel function. We show that the HCN1 subunit interacts both in vivo and in vitro with Nedd4-2 via an extended PY motif in the HCN1 C terminus, resulting in channel ubiquitination. In the brain, HCN1 channels are predominantly expressed in cortical regions (20), but their subcellular distribution differs remarkably between cortical neuronal subtypes (21, 22). Subcellular trafficking may be regulated at the HCN1 C terminus, which has been identified as a target of several binding proteins (12, 14, 23–25). Thus, Nedd4-2 could compete for binding with these proteins to regulate HCN1 subcellular localization and function.

MATERIALS AND METHODS Animals and tissue preparation Ten young adult Wistar rats (30 d old) were used for immunohistochemistry and in vivo coimmunoprecipitation experiments. All animal experiments were performed according to legal guidelines and were approved by the institutional committee for the care and use of laboratory animals (University of Hamburg Animal Care Committee: protocols. ORG_471 and ORG_472). The animals were maintained on a 12-h light-dark cycle at the local animal facility (University of Hamburg Medical Center) and were provided with food and water ad libitum. For immunohistochemistry, 5 of these rats were deeply anesthetized with 12 mg/ml ketamine and 1.6 mg/ml xylazine in saline, i.p. and then transcardially perfused with phosphate-buffered saline (PBS) followed by icecold 4% paraformaldehyde (PFA). The brains were removed, postfixed in 4% PFA (4 h), transferred to 30% sucrose solution (24 – 48 h), and frozen in isopentane (⫺50°C). For in vivo coimmunoprecipitation, the other 5 rats were killed by decapitation. The brains were removed, and the hippocampus, somatosensory cortex, and cerebellum were quickly explanted. The tissue was immediately deep frozen in liquid nitrogen and stored at ⫺80°C until further use. Immunohistochemistry For double-immunolabeling of HCN1 and Nedd4-2, the brains were cut on a cryotome (HM560; Microm, Walldorf, Germany), and sections (30 ␮m) were collected in PBS. Selected sections were preincubated, “free-floating” (26) in 3% bovine serum albumin in PBS for 1 h at room temperature, followed by primary antibody incubation with goat polyclonal anti-HCN1 (sc-19706, 1:600; Santa Cruz Biotechnology, Heidelberg, Germany) and rabbit polyclonal antiNedd4-2 (ab-46521, 1:2000; Abcam, Cambridge, UK) for 48 h 2

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at 4°C. Subsequently, the sections were washed twice for 15 min in PBS, before secondary antibodies were applied (donkey anti-goat-IgG-Alexa Fluor 488 and donkey anti-rabbit-IgGAlexa Fluor 542, 1:600; Life Technologies, Frankfurt, Germany) for 3 h at room temperature. The sections were washed again, treated for 1 min with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Hamburg, Germany), mounted on glass slides, and embedded in fluorescenceprotecting mounting medium (Dako, Hamburg, Germany). For controls, corresponding sections were incubated with each of the primary antibodies alone (single-labeling) or none of these antibodies (negative control), while all other steps (i.e., incubation with both secondary antibodies) were identical. Expected patterns of staining (single labeling) or no labeling (negative control) were the results of this treatment. Sections were viewed and photographed with an Axiophot fluorescence microscope (Leica, Wetzlar, Germany). DNA plasmids and constructs All enzymes were purchased from ThermoScientific (Schwerte, Germany). All oligonucleotides for use in PCR amplification were synthesized by Eurofins MWG Operon (Ebersberg, Germany). The correctness of the cloned constructs and the proper introduction of all deletions were verified by DNA sequencing. Mouse HCN1 cDNA was kindly provided by Drs. Bina Santoro and Steven Siegelbaum (Columbia University, New York, NY, USA). Generation of the hemagglutinin A (HA)-tagged HCN1 (HCN1-HA) construct in pGW1 is described in detail in Lewis et al. (14) For coimmunoprecipitation studies, HCN1 containing a carboxy-terminal myc-tag was used. For this purpose, PCR was used to subclone HCN1 into the expression vector pcDNA6/myc-HisB at the BamHI/EcoRI sites. Deletion mutants were generated using the Phusion Site-Directed Mutagenesis Kit (ThermoScientific), resulting in the following mutant HCN1 constructs: HCN1(⌬610 –700)myc, HCN1(⌬610–910)-myc, HCN1(⌬701–910)-myc, HCN1(⌬801–910)-myc, and HCN1(⌬908 –910)-myc. A Nedd4-2 cDNA–encoding mouse (GenBank accession number BC039746) was generated by PCR, using forward primer 5=-ATGCAAGCTTATGGAGAGACCCTATACATTTAAGGATTTTC-3= and reverse primer 5=-ATGCTCTAGATTAATCCACACCTTCGAAGCCTTGAG-3=, followed by subcloning into the HindIII/XbaI sites of p3x-Flag-CMV. Generation of the construct containing the tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), isoform (1a-4), in pXEGFP is described in detail in Lewis et al. (14). Cell culture Human embryonic kidney 293 (HEK293) cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal calf serum, penicillin, and streptomycin (Sigma-Aldrich). HEK293 cells were maintained at 37°C with 5% CO2. For generation of a stably HCN1-expressing cell line (HEK293-HCN1), the cells were transfected with a pcDNA3-HCN1 construct by using X-tremeGene9 (Roche Diagnostics, Mannheim, Germany). At 24 h after transfection, selection was started with 0.4 mg/ml G418 (Biochrom, Berlin, Germany). After 14 d, the surviving cells were further cultivated and controlled for expression of the HCN1 construct by immunocytochemistry. Immunocytochemistry For confocal microscopy analysis of subcellular HCN1 distribution, HEK293-HCN1 cells were plated on glass coverslips and transfected with vectors coding for either Nedd4-2-Flag

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or TRIP8b(1a-4) or cotransfected with both. The cells were fixed 48 h after transfection for 1 h with 4% PFA, subsequently blocked in PBS supplemented with 5% fetal calf serum, and permeabilized in PBS containing 0.2% Triton X-100. For incubation with the primary antibodies (overnight at 4°C in blocking solution), the following antibodies were coapplied: goat polyclonal anti-HCN1 (1:1000); mouse monoclonal anti-TRIP8b, constant region (1:1200; NeuroMab, Davis, CA, USA); and rabbit polyclonal anti-Flag (1:4000; SigmaAldrich). After several washes with PBS containing 0.1% Triton X-100, the primary antibodies were detected with secondary antibodies: donkey anti-goat-IgG Alexa Fluor 488, donkey anti-mouse-IgG Alexa Fluor 546, and donkey antirabbit-IgG Alexa Fluor 647, respectively (1:900 each; Life Technologies). The cells were counterstained with DAPI, mounted on glass slides, and embedded in fluorescenceprotecting mounting medium. Coimmunoprecipitation (Co-IP) For HCN1-Nedd4-2 Co-IP from tissue (hippocampus, somatosensory cortex, and cerebellum), protein extracts were generated by lysis in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 5 mM EDTA; and Complete protease inhibitor mix; Roche Diagnostics), followed by centrifugation at 13,000 g and 4°C for 30 min. The lysates were further processed as described below. For Co-IP from HEK293 cells, the cells were transfected with the plasmids described above using X-tremeGene9 (Roche Diagnostics) according to the manufacturer’s protocol. Lysis was performed 48 h after transfection in ice-cold lysis buffer containing 500 mM NaCl, to reduce nonspecific binding. The cells were washed twice in PBS, scraped in lysis buffer, and lysed with the aid of a syringe and a 23-gauge needle. The lysates were centrifuged at 13,000 g and 4°C for 10 min and precleared with Protein A or G beads (EZ view; SigmaAldrich). Antibodies were added to the lysates and incubated overnight at 4°C. Protein A or G beads (40 ␮l) were added, and the samples were incubated for another 4 h at 4°C. The beads were precipitated and extensively washed with lysis buffer. The proteins were eluted using SDS-containing sample buffer. HEK293 cell lysates were incubated for 4 h at 4°C with 25 ␮l polyclonal antic-myc agarose (Sigma-Aldrich) or anti-Flag M2 agarose (Stratagene, La Jolla, CA, USA), respectively. The beads were precipitated and washed 6 times with lysis buffer containing 500 mM NaCl. Proteins were eluted into SDS-containing sample buffer and boiled for 5 min, followed by Western blot analysis. Surface expression Surface HA immunoprecipitation was performed according to Lewis et al. (14). Briefly, HEK293 cells were cotransfected with pGW1-HCN1-HA-Ex and pXEGFP-TRIP8b(1a-4), p3xFlag-Nedd4-2, or empty vector containing only Flag (p3xFlag) for the control. At 48 h after transfection, the cells were incubated with a monoclonal anti-HA antibody (Santa Cruz Biotechnology) diluted 1:200 in DMEM at 37°C for 20 min. After incubation, the cells were rinsed twice in warm DMEM, followed by 3 rinses in ice-cold PBS, at which point they were collected and lysed in lysis buffer containing 150 mM NaCl. Protein G beads were used to immunoprecipitate bound anti-HA and were washed 6 times with cold lysis buffer followed by Western blot analysis. Western blot analysis For Western blot analysis, extracts were run on 10% SDSPAGE under denaturating conditions. Samples were boiled UBIQUITINATION OF HCN1 CHANNELS

for 5 min, briefly cooled on ice, and then separated at a voltage that prevented excessive heat. Proteins were blotted on nitrocellulose membranes, and the blots were treated with 5% powdered milk in PBS with 0.3% Tween 20 and incubated with primary antibodies overnight at 4°C. Antibody binding was detected with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA). The following primary antibodies were used: goat polyclonal antiHCN1 (1:250); rabbit polyclonal anti-Nedd4-2 (1:1000); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Life Technologies;); mouse monoclonal anti-TRIP8b, constant region (1:1000); monoclonal mouse anti-c-myc (9E10; 1:10,000; Sigma-Aldrich); monoclonal mouse anti-Flag (M2; 1:5000; Stratagene); monoclonal mouse anti-ubiquitin (P4D1; 1:1000; Cell Signaling, Danvers, MA, USA); and monoclonal mouse anti-ubiquitin (FK1; 1:1000; Merck Millipore). Densitometric analysis of band intensities was performed with ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). Quantification and statistical analysis of HEK293 cell experiments Quantification of Co-IP assays with HCN1 mutants HCN1-myc was precipitated from the HEK293 cells. Coprecipitated Nedd4-2-Flag was then normalized to Nedd4-2-Flag input and presented as a percentage of the control (HCN1 wild-type). Quantification of HCN1-HA expression HA signal from total lysates or from the surface was normalized to GAPDH and presented as a percentage of the control (empty Flag vector). To determine relative surface expression, the surface values were divided by total lysate values and related to the control (set as 100%). Please note that for the inclusion of TRIP8b(1a-4) values, data from experiments comparing TRIP8b(1a-4) and Nedd4-2-Flag (but not the empty Flag vector) were normalized to data from experiments comparing Nedd42-Flag and empty Flag vector. Results for Nedd4-2-Flag were virtually identical in both of these experiments. Analysis of HCN1 N-glycosylation For the analysis of N-glycosylation, the ratio of glycosylated (⬃125-kDa) HCN1 subunits in the channel pool was calculated. Data are presented as the glycosylated percentage of the total surface of HCN1. N-glycosylation of HCN1 was verified using peptide-N-glycosidase-F (PNGase-F; see Supplemental Fig. S1B). Analysis of subcellular HCN1 distribution Slides containing HEK293-HCN1 cells were screened for cells expressing Nedd4-2-Flag, TRIP8b(1a-4), or both, and individual cells (or cell groups) were then photographically captured with an LSM 510 Meta confocal microscope (Zeiss, Jena, Germany) equipped with a Plan-Apochrome ⫻63 oil objective (numerical aperture, 1.4; Zeiss). Equal conditions pertained when capturing cells from different treatment groups. Quantitative analysis was performed with ImageJ. First, cells were selected that had clearly recognizable cell borders and nuclei. For each cell, 3 areas were then marked with the ImageJ selection tool: area 1, the entire cell surface including the plasma membrane; area 2, an area covering ⬃80% of the cell surface; and area 3, the area of the nucleus. Within each area, the integrated density of the HCN1 signal was then measured. Subsequently, density values were sub3

tracted to define signal within the outer (area 1 ⫺ area 2; ⬃20%) and the inner (area 2 ⫺ area 3) portions of the cytoplasm. Values from each area and experimental group were collected (n⫽25 each) and analyzed. Statistical analysis All statistical analyses were performed with ANOVA followed by Bonferroni’s post hoc test (Prism software; GraphPad, San Diego, CA, USA). Data are presented as means ⫾ sem. Values of P ⬍ 0.05 were considered significant. Oocyte injection and electrophysiology Female Xenopus laevis frogs were anesthetized in ethyl 3-aminobenzoate methanesulfonate (1.2 mg/ml tap water; Sigma-Aldrich), part of the ovary lobes was surgically removed, and the tissue was digested for 3–5 h in a calcium-free solution containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and 1.3 mg/ml collagenase A (pH 7.5; Sigma-Aldrich) with NaOH. Defolliculated stage V–VI oocytes were selected the next day, and cRNA was microinjected with a Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA). HCN1 channels were expressed in the absence or presence of Nedd4-2 and/or TRIP8b(1a-4). We used 25 ng HCN1 cRNA, 10 ng TRIP8b(1a-4) cRNA, and either 25 or 100 ng Nedd4-2 cRNA per oocyte. Alternatively, Nedd4-2 and/or TRIP8b(1a-4) cRNAs were replaced by equimolar amounts of EGFP cRNA. Injected oocytes were incubated at 18°C in a solution containing 75 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and 50 ␮g/ml gentamicin (Sigma-Aldrich; pH 7.5 with NaOH), and used for recordings after 3 and 6 d. The currents were recorded at room temperature (20 –22°C) by 2-electrode voltage-clamp with a TurboTec-10CX amplifier (npi, Tamm, Germany) controlled by Pulse software (HEKA Elektronik, Lambrecht, Germany). The oocytes were superfused with a solution containing 94 mM NaCl, 4 mM KCl, 2 mM MgCl2, and 10 mM HEPES (pH 7.5 with NaOH). The holding voltage was ⫺30 mV, and activation was examined with voltage pulses of 3-s duration between ⫹5 and ⫺125 mV (in 10-mV increments). As a measure of channel expression and voltage-dependent channel activation, tail-current amplitudes were measured at 0 mV. Data were analyzed with PulseFit (HEKA) and Kaleidagraph (Synergy Software, Reading, PA, USA). Voltage-dependent fractional activation was analyzed with a Boltzmann function: I/Imax ⫽ 1/1 ⫹ exp [(V ⫺ V1/2)/k]; where I/Imax is the normalized tail-current amplitude, V1/2 is the voltage needed for half-maximum activation, and k is the slope factor. Pooled data are presented as means ⫾ sem. For statistical analysis, tail-current amplitudes and V1/2 values were compared by using ANOVA with the Tukey post hoc test (implemented in Kaleidagraph).

RESULTS Nedd4-2 binds to HCN1 channels via a PY motif and a glutamine-rich region (aa 701– 800) in the C terminus Sequence analysis revealed that HCN1 (rodent and human), but not other HCN subunits, contains a PY motif in its intracellular C terminus (aa 654–657; Fig. 1C, red), suggesting that HCN1 may be a target of Nedd4-2. To probe for this possibility, we first coexpressed Nedd4-2-Flag and HCN1-myc (or the corresponding empty vectors) in HEK293 cells for 48 h and then 4

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immunoprecipitated the tags from the whole-cell lysates, using anti-Flag or anti-myc antibodies. Subsequently, the precipitate was analyzed with the alternate antibody in a Western blot, revealing distinct bands of ⬃125 kDa, indicative of HCN1-myc (Fig. 1A, top panels, arrow) or ⬃130 kDa, indicative of Nedd4-2-Flag coprecipitation (Fig. 1A, bottom panels, arrowhead), if both proteins were coexpressed. No signal (anti-myc) or low background signal (anti-Flag) was detected, if only one of the proteins was present. We then repeated this experiment using anti-myc to precipitate and anti-HCN1 to detect HCN1-myc (Fig. 1B, top panel). Again, Nedd4-2-Flag strongly coprecipitated, when coexpressed with HCN1-myc (Fig. 1B, second panel, arrowhead). (Note that because antiNedd4-2 was used for detection, a weak Nedd4-2immunopositive band, most likely reflecting endogenous Nedd4-2 in the HEK293 cells, is visible in the negative control; Fig. 1B, second panel, asterisk.) Precipitates were further subjected to antibodies recognizing protein-bound ubiquitin. Two different antibodies were used (27): anti-P4D1, recognizing single ubiquitin moieties as well as ubiquitin chains (i.e., mono- and polyubiquitination), and anti-FK1, recognizing only ubiquitin chains (i.e., polyubiquitination; for the positive control of these antibodies, see Supplemental Fig. S1A). Only anti-P4D1 (Fig. 1B, third panel), but not anti-FK1 (Fig. 1B, bottom panel), revealed an increased signal in the precipitate from HEK293 cells that had coexpressed HCN1 and Nedd4-2 (arrow), suggesting that detected proteins, which mostly represent HCN1, have been mono- rather than polyubiquitinated. However, because the ubiquitin signal covers molecular sizes up to ⬃240 kDa, several lysine residues within HCN1 may have been monoubiquitinated, resulting in multiubiquitination. We next wanted to verify the role of the PY motif in Nedd4-2 binding and determine whether other sequences could also contribute to the interaction. For this purpose, various deletion mutants of HCN1-myc (Fig. 1C) were coexpressed with Nedd4-2-Flag in HEK293 cells and subsequently immunoprecipitated. N-terminal deletions did not significantly affect Nedd4-2 binding and were not further pursued (data not shown). At the C terminus, full deletion of the sequence distal to the cyclic nucleotide-binding domain (CNBD; ⌬610 –910), which included the PY motif, virtually abolished binding of Nedd4-2 (reduction to 8⫾1% of control levels; n⫽3 each; P⬍0.001; Fig. 1D, E). Deleting a stretch of 90 aa that comprised the PY motif (⌬610 –700) reduced, but did not fully prevent, Nedd4-2 binding (reduction to 53⫾12%; n⫽3; P⫽0.03; Fig. 1D, E), suggesting that other sequences contribute to Nedd4-2 binding as well. Indeed, a C-terminal deletion, in which the PY motif was preserved (⌬701–910), also reduced binding (to 31⫾7%; n⫽3; P⬍0.01). In contrast, deletions of the final 110 aa (⌬801–910) or of the C-terminal tripeptide SNL (⌬908 –910) did not reduce Nedd4-2 binding (Fig. 1D, E), suggesting that, in addition to the PY motif, the region comprising amino acids 701– 800 is important for HCN1-Nedd4-2 interaction. This region contains a polyglutamine stretch (aa 730 –775; Fig. 1C) flanked by segments rich

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Figure 1. Nedd4-2 interaction with HCN1 in vitro. A) Co-IP (IP) of HCN1-myc and Nedd4-2-Flag from HEK293 cells using anti-myc- or anti-Flag antibodies to precipitate tagged proteins. Note that when the precipitate was screened with the alternate antibody, a distinct signal was detected on the Western blot (WB), if both proteins were coexpressed, indicating coimmunoprecipitation of HCN1-myc (top panels, arrow) or Nedd4-2-Flag (bottom panels, arrowhead), respectively. B) Anti-myc immunoprecipitates (IP) from HEK293 cells that were cotransfected according to scheme. Precipitates were subjected to anti-HCN1 (top panel) or anti-Nedd4-2 (second panel) or to the anti-ubiquitin antibodies anti-P4D1 (third panel) or anti-FK1 (bottom panel). Note that Nedd4-2-Flag coprecipitated strongly (arrowhead) with HCN1-myc, if both proteins were coexpressed. Protein-bound ubiquitin was substantially increased in the precipitate from cells that coexpressed both proteins, if anti-P4D1, which recognizes mono- and polyubiquitination (arrow), but not if anti-FK1, which recognizes only polyubiquitination, was applied. Asterisk indicates immunosignal that is likely to derive from endogenous Nedd4-2-expression. C) HCN1 mutant constructs that were used for identification of the Nedd4-2-binding site in panel D. PY motif is highlighted in red; its amino acid sequence is depicted at top. D) Co-IP (IP) of myc-tagged HCN1 mutant constructs from panel C and Nedd4-2-Flag using anti-myc for precipitation. Top panel: precipitates were screened by Western blot (WB) with anti-Flag for Nedd4-2 coprecipitation. Middle panel: Nedd4-2 input. Bottom panel: screening with anti-myc revealed that all HCN1 mutant constructs were expressed. E) Quantitative analysis of the results in panel D. Precipitated Nedd4-2-Flag was normalized to Nedd4-2-Flag input and is presented as percentage of control (Nedd4-2-Flag coprecipitating with full-length HCN1-myc). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

in amino acids proline, serine, and threonine, which could constitute atypical Nedd4-2 binding motifs (28, 29). It should be further noted that a nonsignificant UBIQUITINATION OF HCN1 CHANNELS

increase of Nedd4-2 binding (up to 160⫾26%; n⫽3; P⫽0.1) that was observed when the C-terminal tripeptide SNL (⌬908 –910) was deleted (Fig. 1D, E) suggests 5

that this sequence mediates a negative effect on Nedd4-2 binding. This possibility was not further examined in the present study. Nedd4-2 interacts with HCN1 channels in vivo Having established that HCN1 and Nedd4-2 interact in vitro, we wanted to know whether such an interaction also plays a role in vivo. To answer this question, we immunoprecipitated Nedd4-2 from brain tissue with high HCN1 content (hippocampus, somatosensory cortex, and cerebellum) and probed for HCN1 coprecipitation. From

each of the selected tissues, substantial amounts of Nedd4-2 were immunoprecipitated, suggesting considerable expression of the protein in these brain regions (Fig. 2A, C, E, right panel, arrowheads). Notably, HCN1 coprecipitated with Nedd4-2 from all of these regions, indicating that Nedd4-2 and HCN1 also interact in vivo (Fig. 2A, C, E, left panel, arrows). To further pinpoint the loci of interaction, perfusion-fixed sections from hippocampus, somatosensory cortex, and cerebellum were coimmunostained for HCN1 and Nedd4-2, and regions of protein expression were determined. This analysis revealed that Nedd4-2 is coexpressed in neurons that ro-

Figure 2. Nedd4-2 interaction with HCN1 in vivo. A, C, E) Co-IP of HCN1 and Nedd4-2 from the hippocampus (A), somatosensory cortex (C), and cerebellum (E). Left panel: anti-Nedd4-2 (Nedd4-2) or random IgGs (IgG) were used to precipitate and anti-HCN1 to detect proteins in the Western blot (WB). Note that HCN1 coprecipitated in each of the selected brain regions (arrows), if anti-Nedd4-2 was used for precipitation. Right panel: application of anti-Nedd4-2 to the precipitate shows that the selected tissues expressed substantial amounts of Nedd4-2 (arrowheads). Inp, input; E, expression. B, D, F) Immunofluorescent colabeling of HCN1 and Nedd4-2 in CA1 of the hippocampus (B), somatosensory cortex (cort; D), and cerebellum (cereb; F), demonstrating that Nedd4-2 was coexpressed in neurons that substantially expressed HCN1: pyramidal cells in the hippocampal CA1 (B), pyramidal cells of cortical layer V (D), and cerebellar Purkinje cells (F). However, on the subcellular level, expression patterns of Nedd4-2 and HCN1 did not substantially overlap. In the pyramidal cells, HCN1 mainly localized to the dendritic compartment (B, D; arrowheads), whereas Nedd4-2 was concentrated in the somata (B, D; arrows). In the Purkinje cells, HCN1 localized to the cell surface in the soma and dendrites (F; arrowheads), whereas Nedd4-2 expression appeared limited to the soma (F; arrows). Note that the intense, triangle-shaped HCN1 immunosignal adjoining the Purkinje cells (F; asterisks) does not reflect HCN1 expression within the Purkinje cells, but represents HCN1 in basket cell axon terminals (20). Similarly, HCN1 immunosignal surrounding CA1 pyramidal cell somata (B; asterisks in insets) may represent presynaptic HCN1 in axon terminals, rather than expression in the pyramidal cells (21). Insets show higher magnifications of pyramidal cell somata in CA1 (B) and layer V (D) of the somatosensory cortex. sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; gcl, granule cell layer; pc, Purkinje cells; ml, molecular layer. Scale bars ⫽ 100 ␮m (B, D); 20 ␮m (F and insets). 6

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bustly express HCN1, such as cornu ammonis 1 (CA1) pyramidal cells of the hippocampus (Fig. 2B), layer V pyramidal cells of the somatosensory cortex (Fig. 2D), and Purkinje cells of the cerebellum (Fig. 2F). However, on the subcellular level, expression patterns did not show much overlap. Thus, Nedd4-2 immunosignal was observed in the somata of pyramidal cells in CA1 of the hippocampus or in layer V of the somatosensory cortex (Fig. 2B, D, arrows), whereas the HCN1 channel subunits were concentrated in the dendrites (Fig. 2B, D, arrowheads; ref. 30). Similarly, in Purkinje cells, Nedd4-2 immunoreactivity was found almost exclusively in the somata (Fig. 2F, arrows), whereas HCN1 distribution extended into the dendrites (Fig. 2F, arrowheads). Thus, due to the marked spatial segregation, interaction of the 2 proteins in vivo may be limited to certain cellular compartments. Reduced Ih after coexpression of HCN1 and Nedd4-2 in Xenopus oocytes We next wanted to understand the functional consequences of the interaction between Nedd4-2 and

HCN1. For these studies, we used the Xenopus oocyte expression system. HCN1 channels were coexpressed with Nedd4-2, in the absence or presence of TRIP8b(1a-4). The latter is known to enhance surface expression of HCN1 channels (13, 14). Xenopus oocytes were microinjected with cRNAs for the respective proteins and incubated for either 3 or 6 d before currents were recorded by 2-electrode voltage clamp (Fig. 3). In all experiments, slowly activating currents with no macroscopic inactivation were recorded during hyperpolarizing voltage pulses (Fig. 3A, B). If only HCN1 was expressed, the tail-current amplitude (Itail) at 0 mV after maximum activation (at ⫺105 mV) had a mean amplitude of 0.48 ⫾ 0.07 ␮A (n⫽11) after 3 d and 0.50 ⫾ 0.04 ␮A (n⫽11) after 6 d of expression (Fig. 3A, C). Coexpression of Nedd4-2 resulted in reduced current amplitudes after 3 d (Itail⫽0.07⫾0.01 ␮A, n⫽12; P⬍0.0001; 7-fold reduction; Fig. 3A, C). This reduction was even more pronounced after 6 d of coexpression (Itail⫽0.04⫾0.004 ␮A, n⫽11; P⬍0.0001; 12-fold reduction; Fig. 3A, C). The observed effect was not the result of a reduced amount of total HCN1 protein, which was

Figure 3. Nedd4-2 coexpression reduces HCN1-mediated Ih in oocytes. A) Representative currents activated by hyperpolarizing voltage jumps to ⫺105 mV, followed by tail currents recorded at 0 mV for each experimental group after 3 d (3d(1), top traces) or 6 d of expression (6d(1), bottom traces). Amount of cRNA per oocyte: HCN1, 25 ng; TRIP8b(1a-4), 10 ng; and Nedd4-2, 25 ng. EGFP cRNA was used to obtain equal amounts of total cRNA per oocyte. In all experiments, a slowly activating current with no macroscopic inactivation, characteristic of Ih, was detected. B) In further experiments, HCN1-mediated Ih in the presence of TRIP8b(1a-4) was measured after 3 d (3d(2), top traces) or 6 d (6d(2), bottom traces) of coexpression with either 25 ng (⫻1, ⫹75 ng EGFP cRNA) or 100 ng (⫻4, no EGFP cRNA) Nedd4-2 cRNA per oocyte. C) Quantitative analysis of Itail amplitudes recorded 3 d (3d(1) and 3d(2)) and 6 d (6d(1) and 6d(2)) after cRNA microinjection. Open bars, no Nedd4-2 coexpression; solid bars, 25 ng Nedd4-2 cRNA/oocyte; gray bars, 100 ng Nedd4-2 cRNA/oocyte. Nedd4-2 coexpression caused a significant reduction of the current in both the absence and presence of TRIP8b(1a-4) (3d(1) and 6d(1)). TRIP8b(1a-4) coexpression alone substantially increased the current amplitudes (consistent with ref. 13). When higher amounts of Nedd4-2 were coexpressed with HCN1 and TRIP8b(1a-4), current reduction was augmented (3d(2) and 6d(2)). *P ⬍ 0.05, **P ⬍ 0.01. D) Activation curves for the 4 experimental groups after 3 d of expression (3d(1)) showing that Nedd4-2 had no effect on the voltage dependence of activation in the presence of TRIP8b(1a-4), but induced a slight shift toward more negative potentials when TRIP8b(1a-4) was absent. This finding may indicate a small modulatory effect of Nedd4-2 binding on channel kinetics. TRIP8b(1a-4) coexpression alone caused a substantial negative shift of the voltage dependence of activation, as expected (13, 15). UBIQUITINATION OF HCN1 CHANNELS

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not significantly altered in the presence of Nedd4-2 (data not shown). As expected, coexpression of HCN1 with TRIP8b(1a-4) caused a robust and persistent increase in current amplitudes (Itail⫽1.76⫾0.20 ␮A after 3 d and 3.76⫾0.12 ␮A after 6 d of expression; Fig. 3A, C). A notable effect of Nedd4-2 was also seen, when HCN1 was coexpressed with TRIP8b(1a-4) (Fig. 3A). Thus, triple expression of all 3 proteins resulted in a significant current suppression compared with expression of HCN1 with TRIP8b(1a-4) alone, after both 3 d (Itail⫽1.22⫾0.12 ␮A, n⫽11, P⫽0.03; 1.4-fold reduction) and 6 d (Itail⫽2.33⫾0.08 ␮A, n⫽11; P⬍0.0001; 1.6-fold reduction) of coexpression (Fig. 3C). These findings indicate that the interaction of HCN1 with Nedd4-2 reduces Ih. Coexpression of TRIP8b(1a-4) reduced, but did not prevent, this effect, suggesting that TRIP8b(1a-4) and Nedd4-2 compete for HCN1 binding. Indeed, if higher amounts of Nedd4-2 cRNA were used (100 instead of 25 ng/oocyte), significantly smaller HCN1/TRIP8b currents were measured, both 3 d (Itail⫽0.92⫾0.07 instead of 1.26⫾0.03 ␮A, n⫽12 for either experiment; P⫽0.01) and 6 d (Itail⫽1.17⫾0.07 instead of 1.85⫾0.08 ␮A, n⫽15 for either experiment; P⬍0.0001) after microinjection (Fig. 3B, C). We further examined whether, similar to TRIP8b(1a-4) (13, 15), Nedd4-2 influences the voltage dependence of activation. As expected, in the absence of Nedd4-2, we observed half-maximum activation at more negative voltages when TRIP8b(1a-4) was coexpressed (V1/2⫽ ⫺64.2⫾1.3 mV, n⫽4, in the absence, and V1/2⫽ ⫺75.1⫾1.0 mV, n⫽4, in the presence of TRIP8b; Fig. 3D). In the presence of TRIP8b(1a-4), no further effect on the voltage dependence of activation was seen when Nedd4-2 was coexpressed (V1/2⫽⫺73.7⫾0.6 mV, n⫽4,

P⫽0.2808). The differences in the absence of TRIP8b(1a-4) (HCN1: V1/2⫽⫺64.2⫾1.3 mV, n⫽4; HCN1⫹ Nedd4-2: V1/2⫽⫺67.8⫾0.6 mV, n⫽4, P⫽0.0402), including shallower curves in the presence of Nedd4-2, may indicate a modulating effect of Nedd4-2 binding on HCN1 channel voltage dependence, or they may be due to extremely small current amplitudes in the presence of Nedd4-2, which were hard to analyze. Nedd4-2 reduces TRIP8b(1a-4)-mediated surface expression of HCN1 channels in HEK293 cells Because the recordings from Xenopus oocytes indicated competing effects of Nedd4-2 and TRIP8b(1a-4) on HCN1 surface expression, we further examined these effects in a HEK293 cell line that stably expressed HCN1 (HEK293-HCN1). These cells were either single or double transfected with vectors coding for Nedd4-2Flag or TRIP8b(1a-4). Subsequently, the cells were photographically captured, and subcellular HCN1 distribution was analyzed as indicated in Fig. 4K (see Materials and Methods). This analysis revealed a significant translocation of HCN1 toward the plasma membrane in cells that overexpressed TRIP8b(1a-4) (Fig. 4D–F, arrows), compared to nontransfected cells (Fig. 4D–F, arrowheads). Thus, the fraction of total HCN1 immunosignal detected in the outer portion of the cell (including the plasma membrane) was significantly enhanced in the presence of TRIP8b(1a-4) (TRIP8b: 34⫾1% vs. nontransfected: 22⫾1%, n⫽25 each P⬍ 0.001; Fig. 4L), whereas the fraction detected in the inner, exclusively cytoplasmic, portion was accordingly reduced (Fig. 4M). In contrast, if Nedd4-2 alone was overexpressed, HCN1 immunosignal was predominantly lo-

Figure 4. Nedd4-2 reduces TRIP8b(1a-4)-mediated surface expression of HCN1 channels in HEK293-HCN1 cells. A–J) Representative confocal images of HEK293-HCN1 cells cotransfected with Nedd4-2 (A–C), TRIP8b(1a-4) (D–F), or Nedd42⫹TRIP8b(1a-4) (G–J). A–C) Transfection of Nedd4-2 (A, pink) did not markedly alter HCN1 distribution (B, green), as nontransfected cell signal (B, C; arrowheads) was predominantly detected in the cytoplasm (B, C; arrows). D–F) In contrast, transfection with TRIP8b(1a-4) (D, red) induced a translocation of HCN1 toward the plasma membrane (E, F; arrows; arrowheads denote nontransfected cells). G–J) If Nedd4-2 (G) and TRIP8b(1a-4) (H) were cotransfected, the TRIP8b(1a-4)effect was reduced, resulting in less HCN1 at the plasma membrane, but a stronger HCN1 presence in the cytoplasm (I, J; arrows). K–N) Quantitative analysis of HCN1 distribution according to the scheme in panel K: the cell surface was divided into an outer portion (⬃20%) including the plasma membrane (L), an inner portion (⬃80%) of the surface minus the nucleus (M), and the nucleus (N), and proportional distribution of HCN1 signal within these areas was calculated for each experimental group (see Materials and Methods). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001. 8

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cated in the inner portion of the cell (Fig. 4A–C, arrows), but no significant difference from nontransfected cells (Fig. 4A–C, arrowheads) was detectable (Fig. 4L, M). However, if Nedd4-2 was coexpressed with TRIP8b(1a-4) (Fig. 4G–J, arrows), a significant reduction of the HCN1 immunosignal located in the outer cellular portion, compared with TRIP8b(1a-4) expression alone (Nedd4-2⫹TRIP8b: 29⫾1%, n⫽25, P⬍0.05; Fig. 4L), was noted, indicating that, concordant with the findings in the oocytes, Nedd4-2 had opposed TRIP8b(1a-4)’s actions. It should be mentioned that much care was taken that analyzed areas did not differ significantly between experimental groups (thus, the outer portion represented on average 21.5, 22.7, 22.4, and 21.6% in nontransfected and Nedd4-2-, TRIP8b(1a4)-, and Nedd4-2⫹TRIP8b(1a-4)-transfected cells, respectively) and that the fraction of HCN1 signal in the nucleus was about the same (⬃7%, presumably representing background levels; Fig. 4N). As an additional measure to evaluate effects of Nedd4-2 interaction with HCN1, we assayed surface HCN1 expression in HEK293 cells by using a vector coding for extracellular HA-tagged HCN1 (14). Cells were cotransfected with HCN1-HA and TRIP8b(1a-4), Nedd4-2-Flag, or an empty Flag vector. Total HCN1-HA expression (Fig. 5A, top panels) did not significantly differ in cells cotransfected with either of these vectors (Fig. 5B). However, surface HCN1-HA expression (Fig. 5A, second panels) was clearly reduced in the presence of Nedd4-2, whereas TRIP8b(1a-4) coexpression had the opposite effect (Fig. 5C, D). N-glycosylation of HCN1 channels may be regulated by Nedd4-2 and TRIP8b(1a-4) We further noticed that both Nedd4-2 and TRIP8b(1a-4) coexpression had affected N-glycosylation of the HAtagged HCN1 subunits. Western blots of rodent HCN1 typically reveal 2 distinct bands of ⬃105 and ⬃125 kDa, indicative of nonglycosylated and N-glycosylated HCN1 channel subunits, respectively (refs. 10, 14; see also Supplemental Fig. S1B). These 2 bands were also observed in our experiments using HA-tagged HCN1 (Fig. 5A, arrows). However, in the Nedd4-2-coexpressing cells the fraction of N-glycosylated subunits in the channel pool (Fig. 5A, solid arrows) was significantly reduced, both in the total (Fig. 5E; Nedd4-2: 5⫾1% vs. control: 9⫾1%, n⫽4 each, P⬍0.05) and in the surface (Fig. 5F; Nedd4-2: 20⫾3% vs. control: 30⫾3%, n⫽4, P⬍0.01) preparations, whereas coexpression of TRIP8b(1a-4) had the opposite effect in the total (TRIP8b: 23⫾2%, n⫽6, P⬍0.001 vs. control), but not in the surface (33⫾1%, n⫽6, P⬎0.05 vs. control) channel pool. Further, the fraction of N-glycosylated channel subunits was higher in the surface than in the total cell preparation in all experimental groups, suggesting enrichment of these isoforms en route to the plasma membrane. To control for nonspecific effects of the experimental model, we also used stably HCN1-expressing HEK293 cells (HEK293-HCN1) for the examination of the effects of Nedd4-2 and TRIP8b(1a-4) on HCN1 N-glycosylation. As in the previous experiments, WestUBIQUITINATION OF HCN1 CHANNELS

ern blots from total lysate of these cells (surface analysis was not feasible in this experimental paradigm) revealed a significant enhancement of HCN1 N-glycosylation if TRIP8b(1a-4) was coexpressed [TRIP8b(1a-4): 55⫾2% glycosylated vs. 36⫾3% in mock-transfected controls, n⫽7 each, P⬍0.001; Fig. 6]. However, if Nedd4-2 was coexpressed, the fraction of N-glycosylated HCN1 was not significantly different from that in the controls (Nedd4-2: 34⫾2 vs. 36⫾3% in mock-transfected controls, n⫽7 each, P⬎0.05; Fig. 6). Thus, whereas these results confirm that N-glycosylation of HCN1 channels is subject to regulation by TRIP8b(1a4), the role of Nedd4-2 in this process remains unclear.

DISCUSSION HCN channels play important functional roles in both the central and the peripheral nervous systems and in the heart (3, 31–33). To ensure proper function, there are several regulatory mechanisms that control activity, expression, and subcellular trafficking of these channels (34). Our current study provides evidence that these mechanisms include Nedd4-2-mediated ubiquitination. Our major findings supporting this conclusion are that Nedd4-2 interacts with HCN1 channel subunits via an extended PY motif in vitro, resulting in HCN1 ubiquitination; Nedd4-2 is expressed in neuronal populations with high HCN1 content and interacts with HCN1 in vivo; Nedd4-2 reduces Ih amplitude when coexpressed with HCN1 in Xenopus oocytes; and Nedd4-2 reduces surface expression of HCN1 channels when coexpressed in HEK293 cells. Our data further provide evidence that Nedd4-2 opposes the actions of TRIP8b(1a-4), an auxiliary subunit of HCN1 that promotes surface expression (13, 14) and, as shown here, N-glycosylation of the channels. Ubiquitination is a post-translational modification that involves the covalent conjugation of ubiquitin, a 76-aa polypeptide, to target proteins. Originally thought to assign misfolded cytosolic proteins for degradation by the 26S proteasome (16), it is now known to regulate physiological processes as diverse as the cell cycle, mRNA transcription, protein synthesis, and subcellular trafficking of membrane proteins (17, 35). In addition, because ubiquitination is a rapid, local, and reversible protein modification, it is highly suitable for activity-dependent modulation of synaptic function (36, 37). The mechanism of ubiquitination involves 3 sequential enzymatic steps (E1–E3). In the final step, an E3 ubiquitin ligase transfers the ubiquitin to either 1 (monoubiquitination) or several lysine residues (multiubiquitination) on the target protein or on ubiquitin molecules that have already been attached to the target protein (polyubiquitination). Moreover, the E3 ligases bind to recognition motifs in the target proteins and thus confer target protein specificity (16, 38). Among the ⬃600 E3 ubiquitin ligases, the Nedd4 family has recently been implicated in the regulation of membrane proteins, including voltage-gated ion channels (19). Nedd4 family members are characterized by a unique domain architecture, consisting of an N-terminal calcium/lipid-binding C2 domain, 2 or 4 trypto9

Figure 5. Effects of Nedd4-2 and TRIP8b(1a-4) on surface expression and N-glycosylation of HA-HCN1 transiently expressed in HEK293 cells. A) Representative Western blots illustrating the results of HA immunoprecipitation from HEK293 cells that were transiently transfected with HCN1-HA and cotransfected with empty Flag vector, Nedd4-2-Flag, or TRIP8b(1a-4). Top panel: HCN1-HA expression in total lysates (HCN1-HA total). Second panel: HCN1-HA exposed on the cell surface (HCN1-HA surface). Third panel: GADPH, used as an internal reference for quantification. Fourth panel: blots indicating Nedd4-2-Flag expression. Fifth panel: blot indicating TRIP8b(1a-4) expression. Note that probing for HCN1 revealed 2 distinct bands indicative of N-glycosylated (⬃125 kDa, solid arrows) and nonglycosylated HCN1 (⬃105 kDa, open arrows). However, patterns differed between the experimental groups, indicating that the ratio of N-glycosylated vs. nonglycosylated HCN1 subunits had been altered (see D, E). Further, note that for quantification (B–E), results for TRIP8b(1a-4) from experiments comparing TRIP8b(1a-4) and Nedd4-2-Flag (A, right panels) were normalized to experiments comparing Nedd4-2-Flag to empty Flag vector (A, left panels). B) Quantitative analysis of HCN1-HA expression in total lysate after cotransfection with Nedd4-2-Flag or TRIP8b(1a-4) did not reveal significant differences compared to cotransfection with empty Flag vector control (Ctl; set as 100%). C, D) In contrast, HCN1-HA surface expression was significantly reduced after cotransfection with Nedd4-2-Flag (Nedd4-2: 58⫾10%; Ctl: 100⫾14%; n⫽4/group; P⬍0.05; C) and showed a strong trend toward reduction, if relative surface expression (surface vs. total lysate) was calculated (Nedd4-2: 66⫾11%; Ctl: 100⫾15%; n⫽4, each; P⫽0.10; D); TRIP8b(1a-4) cotranfection caused a trend in the opposite direction (TRIP8b: 193⫾44%, P⫽0.13 vs. Ctl; D). E, F) Quantitative analysis of the ratio of N-glycosylated vs. nonglycosylated HCN1-HA, showing that coexpression of Nedd4-2-Flag had significantly reduced the fraction of N-glycosylated subunits in the total (Nedd4-2: 5⫾1%; Ctl: 9⫾1%; n⫽4/group; P⬍0.05; E) and the surface (Nedd4-2: 20⫾3%; Ctl: 30⫾3%; n⫽4/group; P⬍0.01; F) channel pool. TRIP8b(1a-4) cotransfection caused an increase in the fraction of N-glycosylated HCN1-HA in the total (TRIP8b: 24⫾1%, n⫽6, P⬍0.001 vs. Ctl; E) but not in the surface (TRIP8b: 33⫾1%, n⫽6, P⬎0.05 vs. Ctl; F) preparation. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

phan-rich WW domains (type I and type IV), and a C-terminal HECT domain that mediates the ubiquitin transfer (38). Target recognition involves the WW domains that interact with proline-rich ligand motifs, such as the PY motif (L/PPxY), which is recognized by type I WW domains. Nedd4-2, in particular, has been shown to interact with ion channels, including the epithelial sodium channel (ENaC; ref. 39), voltage10

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gated sodium (40, 41) and potassium channels (42– 44), and chloride channels (45), via a PY motif. However, a PY motif is not always necessary. For example, ubiquitination of KCNQ2/3 channels by Nedd4-2 persisted even when the PY motif was mutated, suggesting that sequences similar but not identical to the classic PY motif may suffice to mediate Nedd4-2 binding (46). Our observations

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Figure 6. Effects of Nedd4-2 and TRIP8b(1a-4) on N-glycosylation of HCN1 stably expressed in HEK293 cells. A) HEK293 cells stably expressing HCN1 were transiently transfected with plasmids encoding Flag-tagged Nedd4-2 or TRIP8b(1a-4) or were left untransfected (mock treatment). After cell lysis, SDS-PAGE, and transfer of proteins to nitrocellulose membranes, Western blots were probed with antibodies directed against HCN1 (top panel). Arrows indicate the N-glycosylated (solid arrow) and the nonglycosylated (open arrow) proteins. Bottom panel: Western blots showing Flag-tagged Nedd4-2 (lane 2) and TRIP8b(1a-4) (lane 3) coexpression by staining with anti-Nedd4-2- and anti-TRIP8b antibodies. Representative Western blots from independently performed experiments (n⫽7) are shown. Positions of molecular size marker proteins are indicated at left. B) Quantitative analysis of the ratio of N-glycosylated vs. nonglycosylated HCN1, showing that coexpression of TRIP8b(1a-4) produced a significant increase in the N-glycosylated fraction of the channel pool (TRIP8b: 55⫾2% glycosylated vs. 36⫾3% in the mock-transfected controls, n⫽7; ***P⬍0.001). In contrast, coexpression of Nedd4-2 did not cause a significant effect in this experimental paradigm (Nedd4-2: 34⫾2% glycosylated, n⫽7; P⬎0.05 vs. Ctl).

support this suggestion, as deletion of the PY motif in the HCN1 C terminus (aa 654 – 657) did not completely abolish Nedd4-2 binding, which further depended on a region distal to the PY motif (aa 700 – 800). This region contains a polyglutamine stretch (aa 730 –775 in mice) that is flanked by segments rich in the amino acids proline, serine, and threonine. Although currently no specific function can be attributed to the polyglutamine stretch, serines and threonines, if phosphorylated, could provide a binding motif (PXpS/pTP; e.g., aa 776 –779) for the type IV WW domain present in Nedd4-2 (28). Such a mechanism has recently been shown to be responsible for the stress-induced internalization of Nav1.6 channels (29). It should be noted, however, that the extent of the polyglutamine stretch varies significantly between species, being largest in humans (compared to rodents). Therefore, with respect to Nedd4-2 binding, species differences must be considered. Potential role of Nedd4-2-mediated ubiquitination of HCN1 channels What could be the functional role of Nedd4-2 interaction with HCN1 channels? HCN1 channels function at the plasma membrane and, as for other ion channels, the type and number of channels on the surface have to UBIQUITINATION OF HCN1 CHANNELS

be tightly controlled. Regulation begins at the level of mRNA transcription, which can be influenced by environmental factors (47, 48). Surface expression of HCN1 channels is further controlled along the secretory pathway through co- and post-translational modifications, such as glycosylation (10, 49), the association with proteins of the cytoskeleton (23), or auxiliary subunits, such as TRIP8b (12, 13, 14, 22, 50, 51). Once in the plasma membrane, the number of channels is dynamically regulated by means of endocytosis and subsequent retransport to the membrane from endosomes (recycling) or degradation of the channel proteins in lysosomes (52). According to our data, Nedd4-2 contributes to this regulation by reducing the number of HCN1 channels inserted into the plasma membrane. This decrease could occur via 2 mechanisms: first, newly formed HCN1 subunits may be ubiquitinated en route to the plasma membrane while passing through the secretory pathway, resulting in retrotranslocation to the cytoplasm and degradation of the channel subunits in proteasomes. Although this mechanism, generally serving as a quality control for the correct folding of membrane proteins, would reduce the number of HCN1 channels in the plasma membrane, it is less likely to be the major mechanism at work, because the 26S proteasome recognizes only substrates that are conjugated with a polyubiquitin chain containing ⬎4 ubiquitins (16), whereas our data suggest that HCN1 is mono- or multi- rather than polyubiquitinated (Fig. 1B). Alternatively, Nedd4-2-mediated ubiquitination could serve as an internalization signal for channels already present at the plasma membrane, resulting in their endocytosis and subsequent degradation in lysosomes (35, 53). However, experiments using lysosomal inhibitors have so far not supported a specific role of these organelles (unpublished results). In addition, our immunohistochemical data (Fig. 2) did not suggest a preferential localization of Nedd4-2 close to the plasma membrane, which would be expected, if promoting endocytosis were its main function. Interestingly, recent studies have revealed that ubiquitination does not necessarily lead to degradation of membrane proteins, but may also promote their tagging to endosomes or the trans-Golgi complex for further cellular sorting (54, 55). Considering all these possibilities, further studies are necessary to resolve this question. Are Nedd4-2 and TRIP8b(1a-4) competing for HCN1 binding? Noting that the binding site for Nedd4-2 in the HCN1 C terminus overlaps with the binding sites of a recently identified auxiliary subunit of HCN1, the Rab8b-associated protein TRIP8b (24, 25), we further hypothesized that Nedd4-2 and TRIP8b compete for HCN1 binding. To test this hypothesis, we used a TRIP8b isoform (1a-4) that strongly promotes surface expression of HCN1 (13, 14). Several of our findings support the hypothesis. Thus, both in Xenopus oocytes and HEK293 cells, coexpressing Nedd4-2 opposed the effects of TRIP8b(1a-4) on Ih amplitude and HCN1 surface ex11

pression, respectively. Notably, Nedd4-2 effects on Ih amplitude were dose dependent, since the presence of higher amounts of Nedd4-2 led to a stronger suppression of the TRIP8b(1a-4)-mediated increase of Ih in oocytes (Fig. 3). However, in neurons—at least in the cortical pyramidal and cerebellar Purkinje cells that were examined in this study—patterns of Nedd4-2 and TRIP8b (which usually colocalizes with HCN1; refs. 12, 50) did not show much overlap (Fig. 2), suggesting that the in vivo sites of action for TRIP8b and Nedd4-2 are spatially segregated and that competition may thus be less likely. Still, there may be points of intersection. For instance, in CA1 pyramidal cells, in which TRIP8b(1a-4) promotes the trafficking of HCN1 channels from the soma into the distal dendrites (50, 51), TRIP8b binding to HCN1 is decreased under certain conditions (e.g., status epilepticus followed by spontaneous seizures; ref. 56), and the channels are relocated to the somatic compartment, where they could be ubiquitinated and either subsequently degraded (57) or stored for reuse (54, 55). Ubiquitination of HCN1 channels by Nedd4-2 could thus contribute to maintaining homeostasis in neurons by limiting their excitability. As Nedd4-2 activity is modulated by external factors such as stress (58, 59), the neuronal environment can influence this type of regulation. Effects of Nedd4-2 and TRIP8b(1a-4) on HCN1 N-glycosylation Our data further suggest that Nedd4-2 is involved in the regulation of N-glycosylation of HCN1 channels. Generally, N-glycosylation is a co- and post-translational process that promotes proper folding, stability, and oligomeric assembly of ion channels in the endoplasmic reticulum (ER) and facilitates their transport to the plasma membrane (60). Further, once the channels are inserted into the plasma membrane, the electrically charged sugar moieties contribute to the extracellular electric field and can thus influence voltage sensitivity and gating of the channels (61, 62). Therefore, type and extent of ion channel glycosylation have to be controlled. HCN1 subunits contain a single Asn-X-Ser/ Thr consensus sequence for N-glycosylation in the S5–S6 linker. Notably, N-glycosylation is not required for the insertion of functional HCN1 channels into the plasma membrane, because nonglycosylated channel subunits are detected at the cell surface (49, 63, 64), although enrichment of N-glycosylated subunits en route to the plasma membrane occurs (Fig. 5). However, N-glycosylation of HCN1 subunits promotes their heteromerization with subunits of the HCN2 subtype in an activity-dependent manner (10), generating channels with unique physiological properties (65). The contribution of HCN1/HCN2 heteromers to Ih could thus be regulated via controlling the ratio of N-glycosylated vs. nonglycosylated subunits in the physiologically active channel pool. Our data show that this ratio is indeed regulated, because when TRIP8b(1a-4) was coexpressed with HCN1, the fraction of N-glycosylated HCN1 subunits in the channel pool was substantially increased. The precise mechanism and function of this regulation are not yet understood. However, because 12

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TRIP8b is assumed to interact first with HCN1 beyond the Golgi (50), it is unlikely to promote the transfer of N-linked glycans, which is mainly a cotranslational process occurring in the ER, or processing and trimming of the sugar chains, which occur in the ER and the Golgi apparatus (60). Rather, TRIP8b(1a-4) may protect N-glycosylated subunits from deglycosylation or degradation en route to the plasma membrane or during endocytic recycling, thus ensuring that a certain ratio of N-glycosylated vs. nonglycosylated HCN1 channels is maintained at the surface. Further studies are needed for clarification. In contrast to TRIP8b(1a-4), a role of Nedd4-2 in this regulation is less evident. Using an experimental model in which HA-tagged HCN1 and Nedd4-2 were transiently coexpressed in HEK293 cells, we found reduced N-glycosylation in the presence of Nedd4-2 in both the total and the surface channel pool. However, when stably HCN1-expressing HEK293 cells were used, a significant effect of Nedd4-2 was not detectable. The following considerations may help to explain this discrepancy: first, the HA-tag, which did not prevent N-glycosylation in general (as suggested by the glycosylation patterns in Fig. 5), could still have conferred some instability on the N-glycosylated HCN1 protein structure that had promoted Nedd4-2 binding and subsequent degradation; and second, the larger amount of HCN1 channels expressed from transiently compared to stably transfected HEK293 cells could have exhausted the glycosylation machinery, resulting in incompletely glycosylated HCN1 channels that were subsequently targeted by Nedd4-2 and degraded [but protected by TRIP8b(1a-4); see Fig. 5]. The latter possibility, which is further supported by the fact that the fraction of N-glycosylated HCN1 subunits was reduced overall in the transiently compared to the stably HCN1-expressing cells, suggests that Nedd4-2-mediated regulation of HCN1 N-glycosylation is of physiological importance, but its role may be limited to exceptional (e.g., pathologic) situations. Altogether, a role for Nedd4-2 in the regulation of HCN1 N-glycosylation presently remains dubious.

CONCLUSIONS Our findings add Nedd4-2-mediated ubiquitination to a growing list of post-translational modifications and protein interactions involved in the regulation of HCN1 channel trafficking and function (10, 11, 13, 14, 22, 23). In light of increasing evidence for an involvement of HCN channels in neurological and cardiac disorders (31, 32, 33), Nedd4-2 polymorphisms may thus be considered as a cause of HCN1 dysregulation. The authors thank Telse Kock and Christiane SchröderBirkner for excellent technical assistance. This study was supported by Deutsche Forschungsgemeinschaft (DFG; grant: BE4107/2-1).

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Received for publication October 6, 2013. Accepted for publication January 13, 2014.

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