Biochemical Journal

Research article

Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

Laura A. Schrader, Yajun Ren, Feng Cheng, Dui Bui, J. David Sweatt, Anne E. Anderson

Abstract

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

  • cross-talk
  • extracellular-signal-regulated kinase (ERK)
  • mitogen-activated protein kinase (MAPK)
  • phosphorylation
  • potassium channel
  • protein kinase C (PKC)

INTRODUCTION

Regulation of transient outward K+ currents in cardiac myocytes and in neurons can have profound effects on membrane excitability. Kv4 family members are the primary subunits underlying the Ito (transient outward K+ current) in dog and human ventricular myocytes [1], and Kv4.2 is the main subunit underlying this current in rat [2,3] and mouse ventricle myocytes [3,4]. Moreover, in neurons, Kv4.2 is the primary pore-forming subunit of the IA (transient K+ current) in the dendrites of hippocampal pyramidal cells [5], visual-cortical pyramidal neurons [6] and spinal cord dorsal horn neurons [7]. These currents are regulated by kinase activation. IA in the dendrites of pyramidal and dorsal horn neurons is modulated by PKC (protein kinase C) activation [8,9]. In many cell types, including neurons, PKC activates ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) [10], and previous studies have shown that the effect of PKC on IA in area CA1 dendrites is mediated by ERK activation [9,11]. This modulation of IA amplitude can regulate the peak of back-propagating action potentials and excitability of dendrites [5,11,12]. In addition, activation of a number of cell-surface receptors regulates Ito in the heart, possibly through the common effector PKC [13,14]. For example, in lower mammals, Ito of ventricular and atrial myocytes is regulated by α-adrenergic-receptor activation, probably through PKC activation [1518].

PKC activation modulates other voltage-gated ion channels, including Na+ channels [19] and K+ channels [2023]. These PKC effects on K+ channels include the conversion of a rapidly inactivating h-Kv3.4 (human Kv3.4) current into a non-inactivating current [22]. An additional PKC effect is a reduction in currents mediated by both Kv4.2 [23] and L-Kv4.3 (long-Kv4.3), a splice variant of Kv4.3 that has a 19-amino-acid insert that contains a PKC consensus sequence compared with short-Kv4.3 [13,14]. In the case of h-Kv3.4 and L-Kv4.3, these effects are the result of direct phosphorylation of the primary subunit [13,22]; however, the mechanism of the reduction in Kv4.2 current is unknown.

Activation of the PKC pathway can lead to phosphorylation of Kv4.2 channels and modulation of IA via activation of the ERK/MAPK pathway [9,11,24]. We propose that PKC may modulate IA by direct phosphorylation of the Kv4.2 subunit. In the present study, we investigated whether the cytoplasmic regions of Kv4.2 N- and C-termini are directly phosphorylated by PKC and the functional effects of the phosphorylation events.

MATERIALS AND METHODS

Materials

The catalytic subunit of PKC (from rat brain) was obtained from Calbiochem. Glutathione–agarose beads and [γ-32P]ATP were obtained from Amersham Biosciences. The FuGENE™ 6 transfection reagent was obtained from Roche. PDA (phorbol diacetate) was obtained from Sigma.

Synthetic peptides

The PKC-A and PKC-B peptides were synthesized in the Protein Core Chemistry Laboratory at Baylor College of Medicine, Houston, TX, U.S.A.

DNA

The original rat Kv4.2 and human KChIP3 (Kv4 channel-interacting protein 3) cDNAs were generously given by Dr P. J. Pfaffinger (Department of Neuroscience, Baylor College of Medicine, Houston, TX, U.S.A.). Both constructs were in a cytomegalovirus vector. The C- and N-terminal GST (glutathione transferase) fusion proteins were expressed in pGEX vectors.

Phosphorylation of the Kv4.2 GST constructs

The N- and C-terminal cytoplasmic domains of Kv4.2 were expressed in Escherichia coli as GST fusion protein constructs, purified and phosphorylated in vitro following methods described previously [25] except for the following modifications. The recombinant proteins were incubated for 20 min at 37 °C in reaction mixtures (25 μl final volume) containing 20 ng of the catalytic domain of PKC (Calbiochem), Tris buffer 1 [50 mM Tris/HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM Na4P2O7, 10 μg/ml aprotinin and 10 μg/ml leupeptin] and ATP mixture (100 μM ATP, 100 mM MgCl2 and 10 μCi of [γ-32P]ATP). Reactions were stopped by boiling for 5 min with sample buffer. The GST fusion proteins were separated by SDS/PAGE (10% gels) and visualized by Coomassie Brilliant Blue staining. Phosphopeptides were identified by autoradiography. Parallel reactions were performed with GST alone. A time course study of PKC phosphorylation of the Kv4.2 C-terminal peptide was performed.

In the case of phosphorylation of mutant C-terminal constructs, 32P incorporation into mutant and WT (wild-type) proteins was normalized to total protein for each construct. This was then represented as a percentage relative to WT protein (set at 100%).

Phosphorylation of the Kv4.2 C-terminal WT and mutant fusion proteins by ERK (MAP kinase) and PKC was performed as described previously with minor modifications [26]. Briefly, the fusion proteins were incubated for 30 min at 37 °C with 10 μCi of [γ-32P]ATP per reaction in the presence of activated ERK only (as a control) or activated ERK plus PKC (Stratagene), Hepes buffer [25 mM Hepes (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM Na4P2O7, 10 μg/ml aprotinin and 10 μg/ml leupeptin], 10 mM MgCl2 and 100 μM ATP. The reactions were stopped, and samples were resolved by SDS/PAGE and visualized by Coomassie Brilliant Blue staining. Phosphopeptides were identified by autoradiography. To quantify phosphorylation, autoradiographs were analysed further by densitometry using NIH (National Institutes of Health) ImageJ software (http://rsb.info.nih.gov/ij/). 32P incorporation was normalized to the amount of total protein for all constructs, which was then expressed as a percentage of the control (ERK phosphorylation of WT construct), which was set at 100%.

Phosphopeptide mapping

Phosphopeptide mapping was performed following a method described previously [25] with the following modifications. The reaction volume was increased by a factor of 10, the specific radioactivity was increased (20 μCi of [γ-32P]ATP/25 μl reaction volume and 50 μM ATP) and the incubation period was increased to 60 min, based on the time course results. The phosphorylated C-terminal GST-fusion protein was separated by SDS/PAGE (10% gels). The Coomassie Brilliant Blue stained band corresponding to the Kv4.2 C-terminal phosphoprotein was excised and used for phosphopeptide mapping as described previously [25,27] with minor modifications. An in-gel digest with trypsin or endoproteinase Lys-C was performed and, following extraction from the gel, the peptides were separated using reverse-phase HPLC with absorption monitoring at a wavelength of 214 nm. Radioactivity (c.p.m.) in each HPLC fraction was measured as Cerenkov radiation. Phosphopeptides identified as HPLC fractions containing high levels of radioactivity were sequenced using automated Edman degradation. Radioactivity was determined with each sequencing cycle by scintillation counting. It is notable that mapping was first attempted using tandem MS as described previously [28]. However, using electrospray ionization MS (API 3000 LC–MS/MS System; PE Sciex), we were unsuccessful at obtaining the peptide sequences of the Kv4.2 C-terminal domain that contained the candidate PKC phosphorylation sites. Thus we employed the method described above.

Kinetic characterization of the Kv4.2 C-terminal PKC sites

Peptides were synthesized in the Protein Chemistry Core Laboratory (Baylor College of Medicine, Houston, TX, U.S.A.) which contained the PKC phosphorylation sites within the Kv4.2 C-terminal cytoplasmic domain. The synthetic peptides PKC-A (ANAYMQSKRNGLLC) and PKC-B (SRRHKKSFRIPNAC) had a total of 14 amino acid residues corresponding to Kv4.2- channel amino acid residues 441–453 (PKC-A) and 531–543 (PKC-B) within the Kv4.2 sequence. The phosphorylation site was located in the middle (underlined amino acid) of both peptides, and a cysteine residue for coupling to carrier proteins was located at the C-terminus of each peptide.

Kinetic characterization of the PKC phosphorylation sites using the synthetic peptides in kinase assays was performed as described previously [25]. The catalytic domain of PKC (20 ng) was used, and reaction mixtures were incubated at 25 °C. The assays used to determine Km and Vmax were linear with respect to time and linear with added kinase (PKC), and less than 10% of the peptide substrate was converted into product. To obtain the concentration curve for each of the peptides, peptide concentrations ranging from 5–400 μM were used. As a control, parallel reactions were included using the PKC substrate, a synthetic peptide analogue of a fragment of neurogranin [29].

Generation of antisera

The PKC-A and PKC-B synthetic peptides containing the phosphorylated Ser447 and Ser537 PKC sites respectively were coupled to keyhole-limpet haemocyanin and injected into rabbits according to standard protocols (Cocalico Biologicals). The antisera were screened by Western blotting as described previously [25,26] using the phosphorylated and unphosphorylated ovalbumin-coupled synthetic peptides and GST fusion proteins. The antisera were affinity purified against the phosphorylated PKC-A and PKC-B synthetic peptides using Hi-Trap columns (Amersham Biosciences) [25].

Site-directed mutagenesis

The Kv4.2 point mutations were generated using the QuikChange® site-directed mutagenesis kit (Stratagene) as described previously [28,30]. The following primers were used for mutant constructs: S447A Forward, 5′-GCAAATGCCTACATGCAGGCCAAGCGGAATGGGTTAC-3′ and Reverse, 5′-GTAACCCATTCCGCTTGGCCTGCATGTAGGCATTTGC-3′; S447D Forward, 5′-GTGCAAATGCCTACATGCAGGACAAGCGGAATGGGTTACTGAGC-3′ and Reverse, 5′-GCTCAGTAACCCATTCCGCTTGTCCTGCATGTAGGCATTTGCAC-3′; S537A Forward, 5′-CGGAGACACAAGAAGGCTTTCCGAATC-3′ and Reverse, 5′-GATTCGGAAAGCCTTCTTGTGTCTCCG-3′; and S537D Forward, 5′-GCTCACGGAGACACAAAAAAGATTTCCGAATCCCAAATGCC-3′ and Reverse, 5′-GGCATTTGGGATTCGGAAATCTTTTTTGTGTCTCCGTGAGC-3′ (underlining denotes position of mutated nucleotides compared with the original sequence). All mutations were confirmed by DNA sequencing across the entire coding region.

Functional expression in Xenopus oocytes

Oocytes were harvested as described previously [30]. After approx. 24 h, oocytes were injected with 3–10 ng of DNA Kv4.2 (WT or mutant constructs)+KChIP3 (1:1 ratio) using a Nanoject microinjector (Drummond Scientific) into the nucleus of stage V–VI oocytes. Currents were recorded after 2 days by using a two-electrode voltage clamp with an Axoclamp 2A amplifier (Axon Instruments) at room temperature (25 °C). Microelectrodes were pulled from filamented glass (1.5×0.86 mm; A-M Systems) filled with 3 M KCl. The current electrode had a resistance of 0.30–0.50 MΩ, whereas the voltage-electrode resistance ranged from 0.3 to 1.0 MΩ. Currents were leak subtracted online using P/4 leak subtraction. Data were digitized at 2 kHz and stored on a computer equipped with Digidata 1200 software. Current protocols used to obtain data included: (i) activation: hyperpolarization to −110 mV and then depolarization to +40 mV for 400–800 ms, repeated in −5 or −10 mV step intervals; and (ii) inactivation: depolarization to 0 mV and then hyperpolarization to −110 mV for 650 ms, changing this step by +5 mV intervals, then depolarization to 0 mV.

The chamber was continuously perfused with ND-96 buffer [5 mM Hepes/NaOH (pH 7.4), 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 and 1 mM MgCl2] at a rate of 3–6 ml/min. Oocytes expressing mutant and WT (control) DNA were always recorded on the same day and were recorded in at least three batches of oocytes. The data from oocytes expressing WT DNA from different days were not different; therefore all WT data were combined. Data were analysed with Clampfit (pCLAMP sofwtare; Molecular Devices), Origin (OriginLab) and Prism (GraphPad) programs. Peak currents were obtained, and the conductance (G) was determined with a reversal potential (Vrev) of −95 mV according to the equation G=Ip/(VcVrev), where Ip is the peak of the current at a given voltage command (Vc). Activation and inactivation curves were fit with a Boltzmann sigmoidal curve using the equation G/Gmax=1/[1+exp(XV1/2)/slope], where X is equal to the test potential (Vm) and Gmax is the maximum conductance. The mean ±S.E.M. voltage at which half of the currents are activated (V1/2) was determined from the Boltzmann fit and compared among the mutants and WT with a one-way ANOVA and post-hoc test.

Studies in the COS-7 expression system

COS7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin (Invitrogen) in a humidified incubator at 37 °C and 5% CO2/95% air. Cells were maintained in poly-L-lysine-coated six-well plates. Cells were transfected with mammalian expression vectors for Kv4.2 with FuGENE™ 6 transfection reagents following the manufacturer's protocol. Cells expressing Kv4.2 WT and serine-to-alanine and serine-to-aspartate Kv4.2 mutants were stained 48 h after transfection following an immunofluorescence protocol described previously [31]. Briefly, after fixing cells with 4% (w/v) paraformaldehyde for 30 min and incubation in 0.3% Triton X-100 in PBS for 20 min at room temperature, the cells were blocked by incubation with 10% (v/v) fetal bovine serum in PBS for 60 min at room temperature, and then incubated for 60 min at room temperature or overnight at 4 °C with primary antibodies {rabbit polyclonal anti-Kv4.2 antibody (Alomone Labs) generated against amino-acid residues 454–469 (SNQLQSSEDEPAFVSK) of the C-terminal of Kv4.2 or a mouse monoclonal anti-Kv4.2 antibody against an ectodomain [31]}. The Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes) was used for detection of primary antibody binding. Microscopic analysis was performed with a Nikon TE200 inverted fluorescence microscope equipped with a digital camera (Coolmax Fx; RS Photometrics).

Biotinylation of surface proteins

Kv4.2 WT+KChIP3- or AA (Kv4.2 S447A/S537A) mutant+KChIP3-transfected COS7 cells were washed with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (pH 8.0), then treated with sulfo-NHS (N-hydroxysuccinimide)-LC-biotin (0.5 mg/ml; Pierce) at 4 °C for 40 min as described previously [28]. Cells were lysed in RIPA (radioimmune precipitation assay) buffer [150 mM NaCl, 0.5% sodium deoxycholate, 20 mM Na2HPO4, 1% Triton X-100 and 0.1% SDS (pH 7.4)], supplemented with 0.01 mM PMSF, 0.005 μg/ml leupeptin and 0.005 μg/ml pepstatin for 1 h at 4 °C. Lysates were centrifuged at 20000 g for 30 min at 4 °C, and the protein concentration in the supernatants was determined using the BCA (bicinchoninic acid) protein assay kit (Pierce). UltraLink Immobilized NeutrAvidin beads (50 μl; Pierce) were added to each sample, and the mixture was incubated for 1 h at room temperature or 4 °C overnight. The beads were washed four times with cold RIPA buffer and eluted by heating with 50 μl of Laemmli loading buffer (Bio-Rad) for 5 min at 95 °C. The eluates (25 μl) were resolved by SDS/PAGE and immunoblotted with the anti-Kv4.2 antibody (Chemicon). Immunoreactive bands were visualized using a horseradish-peroxidase-conjugated secondary antibody (1:10000 dilution; Cell Signaling Technology). Immunoreactivity values of surface Kv4.2 channels were normalized against levels of actin immunoreactivity in total cell extracts to preclude errors that accompany sample loading and transfer.

Hippocampal slice studies

The N- and C-terminal phospho-selective antibodies were used to evaluate the modulation of PKC phosphorylation of Kv4.2 in rat hippocampal slices. Transverse hippocampal slices were prepared using the method of Roberson et al. [32]. Slices were incubated in ACSF (artificial cerebrospinal fluid) (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2 and 25 mM glucose) at 32 °C for approx. 1 h. Slices were then incubated in vehicle (0.01% DMSO) or 10 μM PDA for 10 min and then slices were rapidly frozen on solid CO2. The slices were subsequently sonicated, normalized according to the BCA protein assay kit and then centrifuged at 60000 rev./min for 20 min at 4 °C using a TLA 120.2 rotor (Beckman), and the pellet was resuspended in a 10% (w/v) SDS solution with 200 mM DTT (dithiothreitol) and protease and phosphatase inhibitors as described previously [25]. After resuspension of the pellet, sample buffer was added. The hippocampal membrane proteins were then used for Western blotting [25,26], with 4–5 slices used for each condition and pooled for sonication.

Data analysis

Immunoreactivity was measured using densitometry (Scion Image). Densitometry data were analysed with a Student's t test, Mann–Whitney test for non-parametric data analysis or with ANOVA and post-hoc test. The GraphPad Prism software package was used for statistical analysis. Results are means±S.E.M. in all cases.

RESULTS

The Kv4.2 amino-acid sequence contains PKC consensus sites

Voltage-gated K+-channel primary subunits are characterized by six transmembrane domains with prominent intracellular N- and C-termini [33]. A schematic diagram of the putative Kv4.2 topology (Figure 1A) and the Kv4.2 amino acid sequence (Figure 1B) are shown. Analysis of the amino acid sequence of Kv4.2 with NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) [34] predicted multiple serine and threonine residues (greater than 50% probability) as candidate PKC phosphorylation sites. Candidate sites for PKC phosphorylation are shown in bold and italic. Four consensus sites for PKC phosphorylation exist in the N-terminal and six consensus sites exist in the C-terminal domains of Kv4.2. The putative consensus PKC sequence is Ser/Thr-X-Arg/Lys, where X is an uncharged residue [35]. The presence of PKC consensus sites within the N- and C-termini suggested that PKC may phosphorylate Kv4.2 at both intracellular termini.

Figure 1 Candidate PKC consensus sites within the Kv4.2 channel subunit

(A) Schematic diagram of the Kv4.2 subunit with a putative tertiary structure of six transmembrane regions, two intracellular loops and cytoplasmic N-terminal and C-terminal domains. The asterisks (*) denote the ten candidate PKC phosphorylation sites in the intracellular domains predicted by the NetPhosK program. (B) The full-length Kv4.2 subunit consists of 630 amino acid residues. The amino acid sequence of the Kv4.2 channel subunit is shown with the six transmembrane-spanning domains underlined. We inspected the primary amino acid sequence of the intracellular domains of the Kv4.2 subunit for known PKC consensus sequences, as these regions may be accessible to intracellular PKC. Four candidate PKC sites on the N-terminal and six candidate sites on the C-terminal are shown (bold and italic font).

The C-terminal of Kv4.2 is a substrate for PKC

To determine if the Kv4.2 N- and C-termini are directly phosphorylated by PKC, Kv4.2 GST fusion proteins of the putative intracellular N- and C-termini were expressed in bacteria and purified. The resulting purified proteins were incubated with PKC and 10 μCi of [γ-32P]ATP in vitro (see the Materials and methods section). Reaction products were separated by SDS/PAGE. Figure 2(A) shows the bands corresponding to the N- and C-termini GST fusion proteins on the Coomassie Brilliant Blue stained gel (left-hand panel, Coomassie). The autoradiograph (left-hand panel, Autorad) revealed 32P incorporation into the C-terminal only, suggesting that the C-terminal, but not the N-terminal, of Kv4.2 was phosphorylated by PKC. In addition, purified GST protein was used as a control and incubated with 32P and PKC. There was no 32P incorporation into GST, indicating that GST was not phosphorylated by PKC (results not shown). In order to determine the optimal time for the saturation of PKC phosphorylation of the Kv4.2 C-terminal substrate, a time course of PKC phosphorylation was performed (Figure 2A, right-hand panel). Saturation of phosphorylation is desirable for mapping the phosphorylation sites in order to be certain that sites are not missed during mapping. Phosphorylation of the Kv4.2 C-terminal reached saturation levels in 40 min. Therefore incubations of 60 min ensured complete phosphorylation of the protein.

Figure 2 PKC phosphorylates the Kv4.2 C-terminal, but not the N-terminal, cytoplasmic domains in vitro

GST fusion-protein constructs of the Kv4.2 N-terminal and C-terminal regions were generated and incubated with the PKC catalytic subunit and [γ-32P]ATP in vitro. Reaction products were separated by SDS/PAGE and visualized by Coomassie Brilliant Blue staining, and 32P incorporation was evaluated with autoradiography. For phosphopeptide mapping, the GST-tagged C-terminal construct was phosphorylated in vitro as described above, and an in-gel digest of the excised band corresponding to the GST-tagged C-terminal construct was performed. This was followed by HPLC and amino acid sequencing of the radiolabelled HPLC fractions. (A) Coomassie Brilliant Blue-stained gels (Coomassie) displayed bands at 54 kDa [GST Kv4.2 C-terminal (CT)] and at 40 kDa [GST Kv4.2 N-terminal (NT)] representing the C-terminal and N-terminal constructs respectively. The autoradiograph (Autorad) shows 32P incorporation into the GST Kv4.2 C-terminal construct only after 30 min incubation, suggesting that only the Kv4.2 C-terminal domain is a PKC substrate. Note that the PKC alone (incubated with all reaction components except peptide substrates) showed no 32P incorporation. A time course of phosphorylation of the GST Kv4.2 C-terminal fusion protein by PKC was performed (right-hand panel). The average normalized attenuance (optical density) of the autoradiograph is shown. By 40 min, the phosphorylation of C-terminal domains reached saturation levels. Results are means±S.E.M. (n=3). (B) The HPLC plot is shown (left-hand panel) with the x-axis representing the time of fraction collection and the y-axis representing the absorbance units (AU) at 214 nm. The radioactivity plot is shown (right-hand panel) with the x-axis representing the HPLC fraction number and the y-axis representing the c.p.m. for β-emission. The radioactivity plot illustrates the peaks in radioactivity that correspond to HPLC fractions 17–19 (black arrow in left-hand panel) and fractions 67 to 68, 69 to 70, 71 to 72 and 73–75 (white arrows in left-hand panel). (C) HPLC fractions 17–19 and 67–75 were sequenced using Edman degradation and the radioactivity released with each sequencing cycle was measured. The sequence of the phosphopeptide in HPLC fractions 17–19 corresponded to amino acids 438–452 within the C-terminal of Kv4.2 (left-hand panel). There was a peak in radioactivity in sequence cycle number 10, indicating that Ser447 was the phosphorylated amino acid (indicated by a circled P above the peptide sequence). The sequence of the phosphopeptide in fractions 67–75 corresponded to amino acids 536–560 within Kv4.2 (right-hand panel). There was a peak in sequence cycle number 2, indicating that Ser537 was the phosphorylated amino acid (indicated by a circled P above the peptide sequence).

Phospho-site mapping of the PKC sites within Kv4.2

To determine the individual PKC phosphorylation sites in the C-terminal of Kv4.2, phosphopeptide mapping and protein sequencing were performed. For these studies, the Kv4.2 C-terminal fusion protein was incubated with PKC in vitro as described above, except that the reaction volume was increased by a factor of 10. The reaction products were resolved by SDS/PAGE. The phosphorylated protein bands were excised, eluted and enzymatically digested. Following digestion, the peptides were separated using reverse-phase HPLC with absorption monitoring at 214, 254 and 280 nm (Figure 2B, left-hand panel). Radioactivity (c.p.m.) in each HPLC fraction was measured as Cerenkov radiation [25]. The phosphopeptide map for the C-terminal construct demonstrated two peaks in radioactivity in HPLC fractions 17–19 and 67–75 (Figure 2B, right-hand panel). HPLC fractions 17–19 (black arrow on HPLC trace) and 67–75 (white arrows on HPLC trace) were sequenced using Edman degradation. Two phosphorylation sites were identified in the C-terminal construct at residues Ser447 and Ser537 (Figure 2C, left-hand and right-hand panels respectively).

Kinetic characterization of the PKC sites was performed using synthetic peptides (see the Materials and methods section) containing the phosphorylation site and six flanking residues on either side. The concentration curves for the PKC Ser537 site revealed that the Kv4.2 Ser537 site was a good substrate for PKC, with a Km of 6.7 μM and a Vmax of 771 nmol/min per mg of protein (mean of two experiments performed in duplicate under steady-state conditions, results not shown). The kinetic characterization for the Ser447 site was not obtained, since there was no 32P incorporation into the synthetic peptide containing this site. A possible explanation for the lack of 32P incorporation into this synthetic peptide is that there are tertiary structural determinants necessary for phosphorylation at this site that are disrupted in the small (14 amino acids) peptide used for the kinetic experiments. The full C-terminal GST fusion protein construct was not used for the kinetic experiments, as it remained on the beads and thus protein concentrations could not be measured accurately, which would be a critical aspect for determining the kinetic values. An additional possible explanation for the lack of phosphorylation in the peptide is that phosphorylation of the Ser537 site may be necessary for phosphorylation at the Ser447 site. Our results from mutating the phosphorylation sites to alanine residues suggest that this, however, is not the case. The Ser447 site is still phosphorylated in the S537A mutant protein (Figure 3).

Figure 3 Mutation of Ser447 and Ser537 to alanine blocks PKC phosphorylation within the K4.2 C-terminal domain in vitro

The Kv4.2 C-terminal cytoplasmic domain contains two predicted PKC consensus sites (Ser447 and Ser537). We used serine-to-alanine mutations (S447A, S537A and AA) within the GST Kv4.2 C-terminal fusion protein to evaluate PKC phosphorylation in vitro. (A) The top panel shows a Coomassie Brilliant Blue-stained gel (Coomassie) with bands at 54 kDa, representing the Kv4.2 C-terminal WT and mutant constructs. The bottom panel represents an autoradiograph (Autorad) of the Coomassie Brilliant Blue-stained gel, which shows 32P incorporation into the WT and the S447A and S537A mutant constructs, but not for the AA mutant construct. (B) The histogram shows the densitometry of 32P incorporation into the WT and mutant GST fusion protein as a percentage of WT incorporation. The single-site mutants did not affect phosphorylation, whereas mutation of both sites to alanine residues (AA) blocked PKC phosphorylation significantly compared with WT control (*P<0.05). Results are means±S.E.M. (n=3), with a one-way ANOVA with post-hoc analysis used for comparison.

Confirmation of phosphorylation sites

These mapped sites were confirmed by site-directed mutagenesis of the C-terminal GST fusion protein. Ser537 and Ser447 of Kv4.2–GST were mutated to alanine residues to block phosphorylation. PKC incubation in vitro with the mutant C-terminal construct in which both Ser537 and Ser447 were mutated to alanine residues (AA) showed no 32P incorporation (Figure 3A), suggesting that these sites were indeed the only sites within the C-terminal that were phosphorylated by PKC (Figure 3B). Furthermore, mutation of the individual sites (S447A alone or S537A alone) showed that both sites were phosphorylated by PKC, and phosphorylation of each site was sufficient to mimic the phosphorylation levels of the WT construct. Interestingly, each site was phosphorylated equally well in the absence of phosphorylation at the other site. These results provide additional support that the C-terminal is indeed phosphorylated at both Ser447 and Ser537 in vitro.

Generation of phospho-site-specific Kv4.2 antibodies

We generated site-selective phospho-specific antibodies to determine if PKC phosphorylates the native Kv4.2 channel in the hippocampus at the sites that had been identified. Two synthetic phosphorylated peptides were produced, corresponding to amino acids A441NAYMQSKRNGLL453 and S531RRHKKSFRI-PNA543 with phosphate groups attached to the appropriate serine residues (Ser447 and Ser537 respectively, underlined). Antisera against the two peptides were generated in separate rabbits. After the terminal bleed, the antisera were affinity-purified using columns with the appropriate peptide. Antibodies were first screened for immunoreactivity to the synthetic peptides (both phosphorylated and unphosphorylated) coupled to ovalbumin for Western blotting. Both the anti-(phospho-PKC-A) antibody (Ser447; Figure 4A, left-hand blot) and the anti-(phospho-PKC-B) antibody (Ser537; Figure 4B, left-hand blot) were specific for the phosphorylated peptide, as there was no recognition of the unphosphorylated peptide. In addition, each antibody was specific for the peptide for which it was generated, and there was no cross-reactivity with the peptide generated for the other phosphorylation site (results not shown). The antibodies were tested against the C-terminal fusion protein. Although the anti-(phospho-PKC-A) antibody was not phospho-specific for the phosphorylated fusion protein (Figure 4A, right-hand panel), the anti-(phospho-PKC-B) antibody was indeed specific for the C-terminal fusion protein only after phosphorylation by PKC, indicating that the anti-(phospho-PKC-B) antibody was phospho-specific (Figure 4B, right-hand panel).

Figure 4 Generation and characterization of the Kv4.2 phospho-specific antibodies for the Ser447 and Ser537 sites

To measure PKC phosphorylation of the Kv4.2 subunits, phospho-selective antisera were generated against phosphorylated PKC-A (phospho-Ser447) and phosphorylated PKC-B (phospho-Ser537) peptides and the affinity-purified antibodies were screened by Western blotting of the unphosphorylated and phosphorylated synthetic peptides and the GST C-terminal Kv4.2 construct (CT Fusion Protein). (A) The peptide sequence (PKC-A peptide, corresponding to amino acid residues 441–453 with an additional cysteine residue at the C-terminus) containing the phosphorylated Ser447 site (indicated by a circled P above the peptide sequence) is shown. The anti-(phospho-PKC-A) antibody showed specific immunoreactivity for phosphorylated PKC-A peptide (left-hand panel), and that immunoreactivity was blocked by pre-incubation of the antibody with the antigen (Ab+Ag, middle panel). The antibody also recognized the phosphorylated and unphosphorylated GST fusion protein, suggesting that the antibody was not phospho-specific for the entire protein (right-hand panel). (B) The peptide sequence (PKC-B peptide, corresponding to amino acid residues 531–543 with an additional cysteine residue at the C-terminal) containing the phosphorylated Ser537 site (indicated by a circled P above the peptide sequence) used for antibody generation is shown. The anti-(phospho-PKC-B) antibody also showed specific immunoreactivity for the phosphorylated peptide (left-hand panel) that was blocked by the antigen (middle panel). This antibody specifically recognized only the phosphorylated GST fusion protein (right-hand panel), suggesting it was a phospho-specific antibody. (C) The phospho-specific antibody against the Ser537 site [anti-(phospho-PKC-B) antibody] was further tested in the hippocampus to determine if it recognized the full-length phosphorylated protein and to determine if Kv4.2 is phosphorylated at Ser537 in the hippocampus. For these studies, acute hippocampal slices were prepared and incubated with vehicle (Control) or PDA (10 μM) for 10 min in vitro. Reactions were stopped by placing slices on solid CO2 and hippocampal membranes were prepared. Hippocampal membrane preparations from vehicle and PDA-treated slices were probed with the anti-(phospho-PKC-B) antibody (S537 phospho-Kv4.2 Ab) (top panel). A histogram of the densitometry results (bottom panel) show that there is a significant increase in immunoreactivity with the anti-(phospho-PKC-B) antibody in response to PDA application to hippocampal slices (n=4–6,*P<0.05). Results are means±S.E.M., and Student's unpaired t test was used for analysis.

The anti-(phospho-PKC-B) antibody was further tested in the hippocampus to determine if it recognized the full-length phosphorylated Kv4.2 protein and to determine if Kv4.2 is phosphorylated at Ser537 in the hippocampus. Rat hippocampal slices were treated with either PDA (10 μM) to stimulate PKC activation or vehicle (Control), and then the membranes were extracted for Western blotting. The anti-(phospho-PKC-B) antibody recognized a band at 69 kDa, suggesting basal phosphorylation of Kv4.2 at that site occurs (Figure 4C, top panel, Control). In addition, the immunoreactivity of the anti-(phospho-PKC-B) antibody increased in response to the addition of PDA [96±31% over vehicle (DMSO), P<0.05 (n=4–6)] (Figure 4C, bottom panel), suggesting the occurrence of increased phosphorylation at Ser537 within Kv4.2 in response to PKC activation.

Functional characterization of PKC sites within Kv4.2

PKC activation causes a reduction in IA in the dendrites of hippocampal pyramidal neurons [11] and dorsal horn neurons [9], and Kv4.2 is the major pore-forming subunit of IA in these neurons [5,7]. Furthermore, activation of PKC with phorbol esters decreases the amplitude of Kv4.2 and Kv4.3 currents in Xenopus oocytes [23]. This could be the result of direct PKC phosphorylation of the channel at the sites that we have mapped. In order to evaluate the functional effects of the PKC phosphorylation sites within Kv4.2, we generated alanine (to block phosphorylation) and aspartate (to mimic phosphorylation) mutant constructs of the phosphorylation sites. We made single- site mutants (S447A or S447D or S537A or S537D) and mutants where both sites were mutated to alanine residues (AA mutant) or aspartate residues [DD (Kv4.2 S447D/S537D) mutant]. These mutant channel constructs and the WT construct were transfected into the COS7 expression system to compare the expression and localization of the channel protein. Immunohistochemistry of COS7 cells expressing the various mutant channel constructs revealed that mutation of Ser447 to alanine or aspartate, Ser537 to alanine or aspartate, or both sites to alanine (AA mutant) or aspartate (DD mutant), residues had no significant effect on channel protein expression (results not shown). However, in order to study the changes in surface localization more specifically, we evaluated the surface expression of the WT and AA mutant channels using surface biotinylation (see the Materials and methods section). We observed that there was a significant increase in the surface expression of the AA mutant compared with the WT construct [61±25% over WT, P=0.028, Mann–Whitney test (n=4)] (Figure 5A). These results suggest that basal phosphorylation of the channel at Ser447 and Ser537 reduces the surface expression of the channel. This reduction in channel surface expression provides a possible mechanism for the reduction in current in response to activation of PKC observed previously [23].

Figure 5 Blockade of phosphorylation at the PKC sites increases the surface expression of Kv4.2 channels in COS-7 cells

Double PKC phosphorylation-site mutant constructs of the full-length Kv4.2 subunit were generated by mutating Ser447 and Ser537 to alanine (AA) or aspartate (DD) and were compared with WT full-length Kv4.2 constructs (WT) when expressed in COS-7 cells (WT and AA co-expressed with KChIP3) or oocytes (AA, DD and WT co-expressed with KChIP3). (A) To evaluate whether phosphorylation at the PKC sites affects the surface expression of the Kv4.2 channel subunits, surface biotinylation was performed in COS-7 cells expressing WT or AA Kv4.2 constructs. Total cell lysates and biotinylated surface proteins of the co-transfected COS-7 cells probed with the anti-Kv4.2 antibody revealed 69 kDa bands, representing that Kv4.2 WT and AA channel proteins were unchanged in total cell lysate (top panel). However, there was a significant increase in the surface expression of the AA mutant construct compared with WT. Untransfected COS-7 cells are shown as a negative control and, as expected, the anti-Kv4.2 antibody did not recognize any protein bands in untransfected cells. Densitometry of surface WT and AA constructs was performed (bottom panel). Surface expression was normalized to Kv4.2 expression levels in total cell lysate. Results are means±S.E.M. (n=4) *P<0.05, with an unpaired Student's t test used for analysis. (B) An example recording of current from WT, AA and DD mutant channels co-expressed with KChIP3 in oocytes is shown. The currents recorded in response to depolarizations from −60 to +40 mV in 20 mV increments are shown. (C) The current/voltage plot of peak current (means±S.E.M.) recorded from oocytes expressing WT+KChIP3 (n=13, ■), AA+KChIP3 (n=8, ▲) and DD+KChIP3 (n=5, ○) is shown. Consistent with the increase in surface expression, the mean peak current recorded from AA+KChIP3 was significantly greater than WT or DD+KChIP3 (P<0.001, one-way repeated measures ANOVA with post-hoc Tukey's test).

Next, we evaluated the effects of mutation of both of these sites (AA or DD mutant channel) on the kinetics of the Kv4.2 current. Recordings were conducted in oocytes expressing the WT and mutant (AA and DD) channels with KChIP3. There was no significant difference in the kinetics of the WT channel control for both the AA and DD mutants, therefore the WT data were combined. Figure 5(B) shows typical current responses to voltage depolarizations from the WT channel and the AA and DD mutant channels. Figure 5(C) is the summary current/voltage curve for the WT and mutant channels. Consistent with the increase in surface expression of the AA mutant channel, the average peak current was significantly greater in the AA mutant channel recordings compared with WT and the DD mutant channels (P<0.001; one-way repeated measures ANOVA with post-hoc Tukey's test). Furthermore, no significant differences in the half-activation voltage (see Table 1; F(2,39)=0.31; P=0.74) or half-inactivation voltage (see Table 1; F(2,27)=0.28; P=0.76) were observed between WT and mutant (AA or DD) channels. Together, these results suggest that surface expression of the AA mutant is enhanced, but the kinetics of the channel is not modulated in the mutant channels.

View this table:
Table 1 Half-activation and half-inactivation voltage of WT and mutant Kv4.2 channels

All constructs were co-expressed with KChIP3 and results are means±S.E.M.

PKC phosphorylation of Kv4.2 increases ERK phosphorylation efficacy in vitro

Interestingly, the Ser537 PKC phosphorylation site lies within a putative ERK-docking domain in the Kv4.2 C-terminal sequence (Figure 6A), which consists of a series of basic amino acid residues [36] corresponding to amino acid residues 532–540 in the Kv4.2 sequence. Because the Ser537 site is localized within this domain, we hypothesized that PKC phosphorylation of the Kv4.2 channel could modulate ERK phosphorylation of the channel. To test this possibility, we evaluated the interaction of PKC and ERK phosphorylation of the Kv4.2 C-terminal fusion protein construct in vitro. The C-terminal GST fusion protein was incubated with activated PKC for 15 min, and subsequently incubated with activated ERK to determine the effect of prior PKC phosphorylation of the C-terminal on ERK phosphorylation. Prior phosphorylation of the C-terminal GST fusion protein by PKC caused a significant increase in phosphorylation of the Kv4.2 C-terminal by ERK (157±47% of PKC/ERK phosphorylation compared with 100±45% of ERK phosphorylation alone, P<0.05) (Figure 6A). This effect was blocked in the S537A mutant (157±47% compared with 72±28%, P<0.05) (Figure 6A) and the AA mutant C-terminal construct (157±47% compared with 42±12%, P<0.01) (Figure 6A). These results suggest that PKC phosphorylation of the C-terminus of Kv4.2 facilitates ERK phosphorylation of this protein and that phosphorylation of the Ser537 site is necessary for this effect.

Figure 6 PKC phosphorylation of the Kv4.2 C-terminal augments ERK phosphorylation of the Kv4.2 C-terminal in vitro

(A) Inspection of the Kv4.2 C-terminal amino acid sequence reveals that a possible ERK docking domain (bold italic underlined residues in Kv4.2 amino-acid residues 532–540) exists on the C-terminal and that the Ser537 site is within that sequence. ERK phosphorylation sites (Thr602, Thr607 and Ser616) are shown (bold and italic residues) in the sequence (top panel). To evaluate the potential interaction of the PKC sites with ERK phosphorylation of Kv4.2, the WT, S537A and AA GST fusion-protein constructs of the Kv4.2 C-terminal domain were incubated first with the catalytic domain of PKC, followed by incubation with activated ERK in vitro. Prior PKC phosphorylation of the WT Kv4.2 C-terminal fusion protein significantly augmented ERK phosphorylation of this construct in vitro (*P<0.05, n=3; bottom panel). This effect was blocked by the S537A (*P<0.05, n=3) and the AA (**P<0.01, n=3) mutant fusion proteins. Results are means±S.E.M. One-way ANOVA with post-hoc analysis was used for comparison. (B) The WT GST C-terminal Kv4.2 construct was incubated without (−) and with (+) PKC, followed by incubation with ERK (+). Western blotting was performed with the ERK single-phospho-site-specific antibodies against Thr602, Thr607 and Ser616 within the Kv4.2 C-terminal domain (top panel). Densitometry of Western blotting (bottom panel) demonstrated that phosphorylation of the C-terminal GST fusion protein by PKC increased immunoreactivity at the ERK Thr602 (T602) phosphorylation site (*P<0.05) and at the Thr607 (T607) site (*P<0.05), whereas the Ser616 (S616) site was unaffected (P>0.05). For these studies, the effect of the WT construct incubated with both PKC and ERK was normalized to that seen for the WT construct incubated with ERK only (WT control). Results are means±S.E.M. (n=3), with an unpaired Student's t test used for analysis.

We have shown previously that ERK phosphorylates the Kv4.2 C-terminal at three amino acid residues, Thr602, Thr607 and Ser616 [26], and that there are functional effects of phosphorylation of these sites [24]. In addition, phospho-specific antibodies for the individual ERK phosphorylation sites were generated. These antibodies were used to determine whether PKC activation affects ERK phosphorylation at one, two or all three of these sites within the Kv4.2 C-terminal GST fusion protein. We found that there was a significant increase in ERK phosphorylation at Thr602 and Thr607, but not at Ser616 in response to prior phosphorylation of the GST fusion protein by PKC (Figure 6B). Densitometry showed that phospho-specific immunoreactivity at the Thr602 [84±46% over WT control (PKC−, ERK+), P<0.05 (n=3)] and the Thr607 [68±24% over WT control (PKC−, ERK+), P<0.05 (n=3)] sites increased significantly, whereas phospho-specific immunoreactivity against the Ser616 site was unaffected (Figure 6B). These results suggest that PKC phosphorylation of the Kv4.2 C-terminus enhances phosphorylation at two of the ERK phosphorylation sites, Thr602 and Thr607, in the Kv4.2 C-terminal.

DISCUSSION

In the present study, we showed that the C-terminal of Kv4.2 is phosphorylated by PKC at two sites, Ser447 and Ser537. Using a phospho-selective antibody against the Ser537 PKC site within Kv4.2, we determined an increase in Kv4.2 phosphorylation at this site following PKC pathway activation in hippocampal slices, indicating that PKC is coupled to Kv4.2 phosphorylation in a system where it is natively expressed. Additional functional results were evident in the COS7 expression system using phospho-site mutants. These studies suggested that PKC phosphorylation of Kv4.2 regulates surface expression of the channel. These studies also showed that when the PKC sites within Kv4.2 were mutated to alanine residues to block phosphorylation, there was a significant increase in surface expression of Kv4.2 compared with WT channels. In addition, PKC phosphorylation of Kv4.2 enhanced ERK phosphorylation of the channel in vitro, suggesting the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

Mimicking or blocking phosphorylation of Kv4.2 at the PKC sites (Ser447 and Ser537) did not modulate the kinetics of the Kv4.2-mediated current, as demonstrated by electrophysiological recordings of the DD and AA Kv4.2 channel mutants. Specifically, there were no differences in the half-activation and half-inactivation voltages between the WT and double-mutant channels (AA and DD). However, as noted above, we demonstrated that surface expression of the AA mutant increased, as indicated by an enhanced peak current in oocytes expressing the AA mutant channel. This suggests that basal phosphorylation of the channel by PKC (or ERK/MAPK) reduces trafficking of the channel to the membrane or maintenance of the channel in the membrane.

We found that prior phosphorylation of the Kv4.2 C-terminal by PKC caused an increase in ERK phosphorylation at two of the three ERK/MAPK sites, Thr602 and Thr607, but not at Ser616 [26]. This is interesting in the context of our previous results; which showed that phosphorylation at Thr602 and Thr607 had an overall inhibitory effect on the current, which mimicked the effect of ERK/MAPK activation seen in the dendrites of CA1 pyramidal neurons [11], whereas phosphorylation at Ser616 caused the opposite effect [24]. The T607D mutation mimicked the effect of ERK seen in neurons, which includes a right-shift in the activation curve and an overall decrease in current. This suggests that the Thr607 site is the relevant site phosphorylated in response to PKC activation in neurons [11]. Moreover, the Thr607 site effect is dominant when all three sites are phosphorylated. Phosphorylation of the C-terminal by PKC prior to ERK may augment phosphorylation at Thr607, increasing the balance of phosphorylation at that site over the Ser616 site to ensure a decrease in current. This does not preclude phosphorylation at Ser616 in response to different physiological stimulation.

Previous results have suggested that filamin is a scaffold protein that links Kv4.2 and the actin cytoskeleton. Furthermore, mutation of amino acids 601–604 in the Kv4.2 C-terminal to alanine residues completely abolished the interaction of Kv4.2 and filamin, as determined by a yeast two-hybrid assay [37]. This suggests that this region is important for the interaction of Kv4.2 with filamin and possibly for Kv4.2 localization and stabilization in the membrane. Phosphorylation of the ERK amino acid residues in this region of the Kv4.2 C-terminal (Thr602 and Thr607) may alter the interaction of filamin and Kv4.2 such that Kv4.2 becomes unstable at the surface membrane. Enhanced phosphorylation of these sites mediated by phosphorylation by PKC may play a role in ‘tagging’ Kv4.2 subunits for internalization. Further studies are necessary to determine the exact role of the interaction of PKC and the ERK phosphorylation sites and the interaction with filamin.

Kv4.2 is the primary subunit that contributes to IA in the dendrites of CA1 pyramidal cells, and the threshold for induction of synaptic plasticity in the CA1 pyramidal cell dendrites is lower in the Kv4.2 knockout animals compared with control [5]. This suggests that a decrease in IA can augment the induction of synaptic plasticity. IA is regulated by activation of PKC, and the effect is blocked by the ERK/MAPK inhibitor U0126 [11]. This suggests the possibility that direct phosphorylation of the Kv4.2 subunit by both PKC and ERK/MAPK may modulate the current. Indeed, our phospho-site-specific antibody demonstrates that Kv4.2 is phosphorylated at Ser537 in the hippocampus in response to PKC activation.

We have shown previously that the Kv4.2 current is modulated by direct phosphorylation of Kv4.2 by ERK/MAPK [24]. These results provide further support for interplay in the regulation of the A-type currents by various kinases and provide another possibility that Kv4.2 is a site of signal integration in neurons and cardiac myocytes. The modulation of ERK phosphorylation of the channel by PKC phosphorylation may be a mechanism of fine-tuning the current in response to a physiological stimulus. Moreover, a greater decrease in current mediated by direct phosphorylation of the channel by both PKC and ERK/MAPK may be a mechanism to regulate dynamically current amplitude and induction of synaptic plasticity.

FUNDING

This work was supported by the National Institutes of Health/National Institute of Mental Health [grant number MH064620] to L. A. S.; the National Institutes of Health/National Institute of Neurological Disorders and Stroke [grant numbers NS039943 (to A. E. A.), NS37444 (to J. D. S.)]; and the Child Neurology Foundation (to A. E. A. and L. A. S.).

Acknowledgments

The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institute of Neurological Disorders and Stroke, the National Institute of Mental Health or the National Institutes of Health.

Abbreviations: AA, Kv4.2 S447A/S537A; BCA, bicinchoninic acid; DD, Kv4.2 S447D/S537D; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; h-Kv3.4, human Kv3.4; IA, transient K+ current; Ito, transient outward K+ current; KChIP3, Kv4 channel-interacting protein 3; L-Kv4.3, long-Kv4.3; MAPK, mitogen-activated protein kinase; PDA, phorbol diacetate; PKC, protein kinase C, RIPA, radioimmune precipitation assay; WT, wild-type

References

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