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Am J Physiol Cell Physiol 290: C852-C861, 2006. First published October 26, 2005; doi:10.1152/ajpcell.00358.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

ERK/MAPK regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit

Laura A. Schrader,1 Shari G. Birnbaum,1 Brian M. Nadin,1 Yajun Ren,2 Duy Bui,1 Anne E. Anderson,1,2 and J. David Sweatt1

1Department of Neuroscience and 2Departments of Neurology and Pediatrics, Baylor College of Medicine, Houston, Texas

Submitted 18 July 2005 ; accepted in final form 19 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Kv4.2 is the primary pore-forming subunit encoding A-type currents in many neurons throughout the nervous system, and it also contributes to the transient outward currents of cardiac myocytes. A-type currents in the dendrites of hippocampal CA1 pyramidal neurons are regulated by activation of ERK/MAPK, and Kv4.2 is the likely pore-forming subunit of that current. We showed previously that Kv4.2 is directly phosphorylated at three sites by ERK/MAPK (T602, T607, and S616). In this study we determined whether direct phosphorylation of Kv4.2 by ERK/MAPK is responsible for the regulation of the A-type current observed in neurons. We made site-directed mutants, changing the phosphosite serine (S) or threonine (T) to aspartate (D) to mimic phosphorylation. We found that the T607D mutation mimicked the electrophysiological changes elicited by ERK/MAPK activation in neurons: a rightward shift of the activation curve and an overall reduction in current compared with wild type (WT). Surprisingly, the S616D mutation caused the opposite effect, a leftward shift in the activation voltage. K+ channel-interacting protein (KChIP)3 ancillary subunit coexpression with Kv4.2 was necessary for the T607D effect, as the T607D mutant when expressed in the absence of KChIP3 was not different from WT Kv4.2. These data suggest that direct phosphorylation of Kv4.2 at T607 is involved in the dynamic regulation of the channel function by ERK/MAPK and an interaction of the primary subunit with KChIP is also necessary for this effect. Overall these studies provide new insights into the structure-function relationships for MAPK regulation of membrane ion channels.

K+ channel-interacting protein; kinase; neurons; A-type current

MANY STUDIES HAVE SHOWN THAT ERK is important for regulation of neuronal function, particularly playing a role in the regulation of synaptic plasticity and long-term memory formation (3, 13, 14, 30, 46, 48). Considerable evidence is accumulating that ERK activation plays a role in the regulation of postsynaptic excitability, specifically operating in the context of synaptic plasticity (40, 46, 48). One potential mechanism of this regulation by ERK is indirect, by long-term modulation of cell properties through the control of gene transcription and regulation of channel gene expression (9). Another possible mechanism by which ERK might modulate neuronal excitability is through direct regulation of membrane ion channels that regulate the membrane potential and thereby intrinsic membrane properties.

In our recent studies, we have focused on regulation of ion channels by ERK because modulation of excitability may be a critical factor that ultimately controls the induction of long-lasting changes in synaptic strength. One possible direct target of ERK is the K+ channel Kv4.2, which encodes a transient A-type K+ current that is present in the dendrites of CA1 pyramidal neurons. Because these currents are at high density in dendrites where the neurons receive synaptic input, rapid voltage-dependent activation of these channels can limit the peak amplitude of back-propagating action potentials as well as modulate incoming synaptic information. Thus these currents can exert profound effects on hippocampal network communication (23). Recent studies indicate that ERK activation can regulate the dendritic A-type K+ currents (15, 47, 51). For example, the amplitude of A-type K+ currents in neurons is decreased by PKA and PKC activation, an effect that is mediated by ERK activation (21, 22, 25, 47, 51). This ERK modulation of A-type current amplitude can enhance the peak amplitude of back-propagating action potentials in the dendrites (22, 51) and thereby enhance the depolarization seen at synapses. Via this mechanism ERK can ultimately, albeit indirectly, control voltage-dependent N-methyl-D-aspartate receptor activation.

There is now good evidence that Kv4.2 is the channel-forming primary subunit that underlies this transient A-type current characterized in pyramidal neurons of the hippocampus, as well as in other brain areas and in cardiac myocytes (17–19, 39, 41, 50). Studies show that Kv4.2 is localized to the pyramidal cell dendrites (29, 37, 42). Furthermore, physiological studies demonstrate that the pharmacological and kinetic properties of Kv4.2-encoded currents in expression systems are similar to the transient outward currents in dendrites (8, 23, 34, 36). In addition, Kv4.2-encoded currents become even more similar in their characteristics compared with the endogenous A-type current when Kv4.2 is coexpressed with other interacting subunits such as K+ channel-interacting proteins (KChIPs) (2, 24, 33) as well as the dipeptidyl aminopeptidase-related protein DPPX (27, 31). Finally, preliminary studies indicate that Kv4.2-knockout animals show a greatly reduced dendritic A-type current in pyramidal cell dendrites (50).

In prior studies (1) we showed that the cytoplasmic carboxy terminus of Kv4.2 is phosphorylated by the ERK subtype of the MAPKs. In these earlier studies we identified three ERK/MAPK phosphorylation sites at amino acid residues T602, T607, and S616. In the present studies we tested the hypothesis that ERK regulates the Kv4.2 A-type K+ channel via direct phosphorylation of the pore-forming {alpha}-subunit at the three sites previously mapped. We used a site-directed mutagenesis approach to determine whether mimicking phosphorylation of Kv4.2 at these sites alters channel functional properties, biophysical characteristics, and cell surface expression.

Overall, our results support the hypothesis that ERK regulates Kv4.2 channels by direct phosphorylation. Our mutagenesis and functional studies indicate that introduction of a negative charge at T607 in the carboxy terminus of Kv4.2 affects the voltage dependence of channel activation and the rate of recovery from inactivation. These effects are reminiscent of the effects of ERK activation on A-type current in intact neurons. This regulation of the outward K+ current is due to changes in gating kinetics of the current, as ERK phosphorylation has no effect on protein expression or surface membrane localization. These results provide a bridge between prior biochemical results obtained in vitro (1) and functional studies performed in the dendrites of neurons and indicate that direct ERK phosphorylation of Kv4.2 channel is a primary mechanism for the ERK-mediated decrease in A-type currents previously reported (21, 22, 47, 51).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Functional expression in Xenopus oocytes. Oocytes were harvested as described previously (33). After ~24 h, oocytes were injected with 3–10 ng of DNA Kv4.2 [wild type (WT) or mutants] + KChIP3 in a 1-to-1 ratio or Kv4.2 (WT or mutants) + green fluorescent protein (1:1), using a Nanoject microinjector (Drummond Scientific), into the nucleus of stage V–VI oocytes. Currents were recorded after 2 days under two-electrode voltage clamp with an Axoclamp 2A amplifier (Axon Instruments) at room temperature. Microelectrodes were pulled from filamented glass (1.5 mm x 0.86 mm; A-M Systems) filled with 3 M KCl. The current electrode had resistance of 0.30–0.50 M{Omega}, whereas the voltage electrode ranged from 0.3 to 1.0 M{Omega}. Currents were leak subtracted online with 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 1) activation: hyperpolarization to –110 mV and then depolarization to +40 mV for 400–800 ms, repeated in –5- or –10-mV step intervals; 2) 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, 3) recovery from inactivation for oocytes expressing Kv4.2 alone: a two-pulse protocol that included a 500-ms depolarization to 0 mV, followed by a hyperpolarization to –110 mV of varying durations (5 ms with subsequently longer hyperpolarizations increasing in increments of 100 ms) for a final hyperpolarization of 705 ms and then depolarization to 0 mV. Kv4.2 + KChIP3 recovered from inactivation more quickly than Kv4.2 alone, so the initial hyperpolarization pulse was 2 ms, increasing in increments of 10 ms for a final duration of hyperpolarization of 192 ms.

The chamber was continuously perfused at a rate of 3–6 ml/min with ND-96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES pH 7.4 with NaOH). Oocytes expressing mutant DNA 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 analyzed with Clampfit, Origin, and Prism programs. Inactivation time constants were fit in Clampex with the Simplex method. Peak currents were obtained and conductance (G) was determined with a reversal potential (Vrev) of –95 mV according to the equation G = Ip/(Vc – Vrev), 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 with the equation G/Gmax = 1/[1 + exp(X – V1/2)/slope], where X is equal to the test potential (Vm) and Gmax is maximum conductance. The mean ± SE voltage at which half 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 Bonferroni test.

DNA preparation and site-directed mutagenesis. The original Kv4.2 and KChIP3 cDNA were provided by Dr. Paul Pfaffinger (Baylor College of Medicine, Houston, TX). Both constructs are in a cytomegalovirus (CMV) vector. Point mutations were made with a site-directed mutagenesis kit (Stratagene). The primers (upper sequence) used included 5'-gcattccagcacctccagtaaccaccccag-3' (T602A), 5'-gcattccagatcctccagtaacc-3' (T602D), 5'-cctccagtaaccgccccagaag-3' (T607A), 5'-cctccagtaaccgacccagaag-3' (T607D), 5'-gacaggcccgaggatcctgagtattccgga-3' (S616A), and 5'-gacaggcccgaggctcctgagtattccgga-3' (S616D). Mutations were confirmed by restriction enzyme digestion and DNA sequencing. In most cases, the entire Kv4.2 region was sequenced to ensure that no other mutations existed.

Expression in COS-7 cells. The FuGene 6 transfection reagent (Roche) was used for COS-7 cells transfections with plasmid DNA of WT Kv4.2 or mutant DNA and KChIP3 (1:1, 2.0 µg of total DNA). Transfected cells were grown on 35-mm plates to a 2 x 105 cell density. Approximately 24 h after transfection, the cells were harvested by scraping in 1 ml of homogenization buffer (HB) containing (in mM) 20 Tris, pH 7.5, 1 EGTA, 1 EDTA, 4 p-nitrophenylphosphate (PNPP); 1 Na3VO4, 100 PMSF, and 1 Na4P2O7, with protease inhibitor cocktail added (1:100; Sigma) and centrifuged at 10,000 rpm for 5 min at 4°C. The cell pellet was resuspended in 500 µl of HB, sonicated, and centrifuged (60,000 rpm for 20 min at 4°C), and the supernatant was removed. The membrane pellet was resuspended in 5% SDS-HB with 100 mM DTT and protease inhibitor cocktail (1:100; Sigma). Sample buffer was then added, and the samples were loaded onto a SDS-PAGE gel (10%) for Western blot analysis (see below).

Surface biotinylation. Transfected COS-7 cells were rinsed twice with cold PBS (pH 7.2), followed by a rinse with cold PBS, pH 8.0, with 1 mM CaCl2 and 1 mM MgCl2. The cells were then incubated with 200 µl of 0.5 mg/ml EZ-Link sulfo-NHS-LC-biotin (in PBS at pH 8; Pierce) at 4°C for 30 min. The cells were washed with cold PBS twice and incubated with 100 mM glycine and PBS at 4°C for 30 min to quench the biotinylation reaction. Cells were then washed three times in PBS. Cells were lysed with 250 µl of RIPA buffer containing (in mM) 150 NaCl, 10 Tris pH 7.2, 5 EDTA, 0.1% SDS, 1% Triton X-100, and 1% deoxycholate, with phosphatase and protease inhibitor cocktail added in each well and constant shaking for 1 h at 4°C. Lysates were then centrifuged at 20,000 g for 30 min at room temperature. The supernatant (150 µl) was incubated in a 1:1 slurry of beads (Ultralink Neutravidin; Pierce) for 2 h at room temperature with RIPA buffer. The pellet was eluted with 50 µl of Laemmli loading buffer and boiled at 100°C for 3 min, and the samples were loaded onto a SDS-PAGE gel. The biotinylated Kv4.2 protein was normalized to the total Kv4.2 assayed from the total cell lysate and then normalized to WT surface expression.

Western blot analysis. Membrane proteins were loaded and run on a 10% acrylamide gel to resolve Kv4.2. Gels were then blotted electrophoretically onto Immobilon membrane paper in a transfer tank maintained at 4°C, with typical parameters being an overnight transfer at a constant current of 250 mA (transfer buffer: 192 mM glycine, 25 mM Tris, and 20% methanol, pH 8.3). Immobilon membranes were blocked for 1 h at room temperature in either 10 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 (TTBS) with 5% powdered milk and 3% BSA (+0.05% microcystin for phosphoantibodies) for Kv4.2 antibodies or TTBS + 3% BSA for ERK antibodies. Primary antibody concentrations were as follows: 1:500 for Kv4.2 polyclonal antibody (Chemicon, Temecula, CA) and 1:500 for 304 phospho-Kv4.2 antibody.

Antibody detection. Immobilon membranes were incubated sequentially with primary antibody and a biotin-labeled goat anti-rabbit IgG secondary antiserum (Cell Signaling; 1:20,000) and then developed with enhanced chemiluminescence (Amersham Biosciences). Blots were washed extensively in 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 after incubations with primary and secondary antibodies (typically 4 washes of 10 min each).

Alkaline phosphatase treatment. Entire mouse hippocampus or COS-7 cells transfected with Kv4.2 + KChIP3 were homogenized in 500 µl of HB containing (in mM) 20 Tris, pH 7.5, 1 EGTA, 1 EDTA, 1 Na4P2O7, 4 PNPP, 1 Na3VO4, 100 PMSF, and microcystin (1:100; protease inhibitor cocktail added). Hippocampi (or COS-7 cells) to be treated with alkaline phosphatase were homogenized in buffer without phosphatase inhibitors [PNPP (Sigma), Na3VO4, microcystin]. Homogenate was treated with 1 or 3 units of alkaline phosphatase (2 units for COS-7 cells) and incubated for 15 min at 30°C. After treatment, homogenates were centrifuged at 1,000 g at 4°C for 5 min. The supernatant was then centrifuged at 60,000 rpm at 4°C for 20 min. The pellet was resuspended in 5% SDS-HB with 200 mM DTT with protease inhibitor cocktail, PMSF, and microcystin. Protein was normalized with a Bio-Rad DC protein assay kit and 4x sample buffer containing (in mM) 3 Tris, pH 6.8, and 200 DTT, with 40% glycerol, 8% SDS, and 0.08 mg/ml bromphenol blue added.

Protein expression and purification. The WT and triple A mutant Kv4.2 carboxy-terminal proteins were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins, using methods modified from Hakes and Dixon (20). Plasmids containing the Kv4.2 carboxy-terminal cDNA were constructed with the GST-fusion vector pGEX-KN (20). A single colony of BL21(DE3)-pLysS cells transformed with the protein plasmid was grown in Luria broth (LB; 170 mM NaCl, pH 7.5, 1% tryptone, 0.5% yeast extract) containing 20 µg/ml carbenicillin and then used to seed a 500-ml culture. After growing to an optical density of 0.6–0.8 (absorbance at 600 nm), the culture was centrifuged (Beckman model J2-21M; 1,000 g, 15 min, 4°C). The cell pellet was resuspended in 500 ml of LB with carbenicillin. The bacteria were induced by incubation at room temperature with 200 µM isopropyl beta-D-1-thiogalactopyranoside (IPTG) for 4 h and harvested by centrifugation.

The cells were resuspended and incubated in Tris buffer 1 (50 mM Tris·HCl, pH 8.0, 2 mM EDTA, 10 mg/ml pepstatin, 10 µg/ml leupeptin, and 100 µM PMSF) containing 10 mM beta-mercaptoethanol and 100 µg/ml of lysozyme (Sigma) for 15 min at 30°C. After solubilization with 1.5% N-laurylsarcosine, the lysate was incubated with 20 µg/ml DNase I (Boehringer Mannheim) and 10 mM MgCl2. The lysate was then centrifuged (Sorvall RT 6000B; 1,000 g, 15 min, 4°C) and adjusted to a 2% Triton X-100 concentration.

The GST-fusion proteins were purified with glutathione affinity absorption. Glutathione agarose beads were washed, resuspended in Tris buffer 1, and then incubated with the lysate for 1 h at 4°C. The beads were washed three times with Tris buffer 1 by repeated centrifugation (Sorvall; 100 g, 5 min, 4°C). After the final wash, the bead preparation was resuspended in Tris buffer 2 (20 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).

Protein phosphorylation. WT and triple A mutant GST fusion proteins were phosphorylated with activated ERK2 for 0.5–1 h at 37°C according to the manufacturer's directions (Stratagene). The reaction included 1.0 µCi/µl reaction of [{gamma}-32P]ATP in the presence of activated ERK2, HEPES buffer (in mM: 25 HEPES, 0.5 EDTA, 0.5 EGTA, and 1 Na4P2O7, with 10 µg/ml aprotinin and 10 µg/ml leupeptin), 10 mM MgCl2, and 100 µM ATP. The reaction was stopped with sample buffer (in mM: 30 Tris·HCl, pH 6.8, and 200 DTT, with 40% glycerol, 8% SDS, and 0.04 mg/ml bromphenol blue), and the samples were boiled for 5 min. The fusion proteins were separated with 10% SDS-PAGE and visualized by Coomassie blue staining. The phosphopeptides were identified by autoradiography.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We previously identified (1) three candidate ERK phosphorylation sites in the carboxy-terminal domain of Kv4.2 through in vitro studies (Fig. 1). Two other ERK consensus sequences exist within the Kv4.2 carboxy-terminal sequence, but these sites were not phosphorylated in our initial studies. We also developed a phosphospecific antibody that recognizes full-length Kv4.2 when it is phosphorylated at all three ERK phosphorylation sites (1). Using this affinity-purified antiserum, we observed that basal levels of phosphorylation of Kv4.2 exist in mouse hippocampal tissue (Fig. 1C) and COS-7 cells (Fig. 1D), confirming that these sites are indeed sites of phosphorylation in the intact Kv4.2 channel in both COS-7 cells and hippocampus.


Figure 1
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  		Fig. 1. K+ channel primary subunit Kv4.2 is phosphorylated at 3 sites by ERK/MAPK. A: schematic diagram of Kv4.2 with 6 transmembrane-spanning domains and amino and carboxy termini. K+ channel-interacting protein (KChIP) is shown interacting with the amino terminus of Kv4.2. B: amino acid sequence of the Kv4.2 subunit. The 3 ERK sites that we previously reported (T602, T607, and S616) are represented by black asterisks in A and by boldface in the amino acid sequence. Two other ERK/MAPK consensus sites exist within the Kv4.2 sequence, S516 and T567, represented by gray asterisks in A. C: basal ERK phosphorylation of Kv4.2 in the hippocampus. Top: a phospho-specific antibody directed against all 3 mapped phosphorylation sites was generated. This antibody recognizes a specific band at the level of Kv4.2 (~69 kDa) in membrane preparations from the hippocampus and is phosphospecific. Hippocampal homogenates were treated with increasing concentrations of alkaline phosphatase (Alk Phos) to dephosphorylate the protein (see METHODS). The immunoreactivity of the phospho-Kv4.2 antibody decreases with increasing alkaline phosphatase concentrations. Immunoreactivity is absent at 3 U of alkaline phosphatase. D: basal ERK phosphorylation of Kv4.2 in COS-7 cells. COS-7 cells were transfected with Kv4.2 and KChIP3 or with no DNA as a control (untransfected). Cell homogenates were treated with 2 units of alkaline phosphatase. Alkaline phosphatase decreased immunoreactivity of a specific band by the phospho-Kv4.2 antibody (top).

 
Given an ERK-mediated decrease in the A-type current in hippocampal slices (47, 51), we hypothesized that phosphorylation of Kv4.2 at these three phosphorylation sites (T602, T607, and S616) would cause a decrease in Kv4.2 channel function. To determine whether direct phosphorylation of Kv4.2 at the ERK sites is responsible for ERK modulation of Kv4.2 function, we chose to construct site mutants of each phosphorylation site to study the effects of phosphorylation at each individual site, as direct ERK activation in cells is difficult. We used aspartate (D) site mutants of the three ERK phosphorylation sites to mimic Kv4.2 phosphorylation. As controls we used both the WT channel and mutants that had the phosphorylation sites mutated to an alanine (A). For both the biophysical and protein surface expression studies, all mutants and WT Kv4.2 constructs were coexpressed with KChIP3 (unless otherwise indicated; see Fig. 5), a modulatory subunit that we and others have shown to affect the biophysical properties and expression levels of Kv4.2. The coexpression of Kv4.2 + KChIP3 in expression systems such as oocytes and COS-7 cells better approximates the functional properties of the native channels (2, 24, 31, 33).


Figure 5
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  		Fig. 5. Coexpression of KChIP3 with the T607 mutant is necessary to manifest effects on activation voltage and recovery from inactivation. The T607D and T607A mutants and WT were expressed alone (without KChIP3, with control GFP) and compared with +KChIP3 expression. A: conductance-voltage curve for WT + KChIP3 and WT – KChIP3. B: conductance-voltage curve for T607A + KChIP3 and T607A – KChIP3. C: conductance-voltage curve for T607D + KChIP3 and T607D – KChIP3. Although +KChIP3 coexpression caused a leftward shift of the activation voltage for the WT and T607A constructs, there was no shift for the T607D mutant, demonstrating that the activation curve of T607D + KChIP3 was similar to that of T607D alone. The mean V1/2 of the T607A, T607D, and WT mutants of Kv4.2 expressed in the absence of KChIP3 were not significantly different (P > 0.05; 1-way ANOVA and post hoc Bonferroni test).

 
Mutation of all three sites to aspartate mimics the effect seen in neurons. In our first experiment we tested the effects of mimicking ERK phosphorylation of Kv4.2 by mutating the T602, T607, and S616 amino acid side chains to D. A wide variety of previous studies, including studies of Kv4.2, have indicated that D mutations can mimic the effects of serine or threonine phosphorylation (6, 11, 45). We found that, indeed, mutation of all three sites to an aspartate (triple D mutant: T602D, T607D, S616D) decreased the transient outward K+ current (Fig. 2). Specifically, the triple D mutant displayed a more depolarized activation voltage compared with WT (Fig. 2B). The activation curve of the triple D mutant was shifted 17 mV to the right toward more depolarized membrane potentials. The V1/2 for the triple D mutant was –7 ± 2 mV, which was significantly different from WT (–24 ± 3 mV; P < 0.01) and the triple A mutant (–26 ± 3 mV; P < 0.01). The inactivation curve (at +0 mV) was fit with a single exponential, and we determined that the time constant of inactivation of the triple D mutant (35 ± 5 ms) was significantly different (P < 0.05) from WT (57 ± 5 ms). Steady-state inactivation of the triple D mutant was not different from WT (Table 1). Although there was a trend toward a slower rate of recovery from inactivation for the triple D mutant, it was not significantly different from WT (Table 1).


Figure 2
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  		Fig. 2. Mutation of all 3 sites to an aspartate to mimic phosphorylation (triple D mutant) causes a shift in the activation voltage compared with wild-type (WT) and triple A mutation. The WT and triple A and triple D mutants were expressed with KChIP3 and compared with WT + KChIP3. A: example of outward K+ current traces from a WT (black) and a triple D mutant (gray) recorded on the same day from the same batch of oocytes. Right: the maximal outward current from the triple D mutant is scaled up to show the change in activation at lower membrane potentials. B: example of outward K+ current traces from a WT (black) and a triple A mutant (gray) recorded on the same day from the same batch of oocytes. Right: the maximal outward current from the WT is scaled up to the amplitude of the triple A mutant to show that there is no effect on activation. The voltage protocol used to obtain the traces is shown below the raw data. The traces are truncated, similarly to those shown in A. C: activation curve showing the normalized conductance vs. test voltage of the WT vs. triple D and triple A mutants. The activation curve of the triple D mutant is shifted toward more depolarized membrane potentials, whereas the activation curve of the triple A mutant is not different from WT. The slope of the activation curve for WT (18 ± 0.7) was not significantly different from the triple D (18 ± 1) or triple A (18 ± 1) mutant. Inset: mean ± SE voltage at which the current was activated (V1/2; acquired from a Boltzman fit of the curve) of WT and triple A and triple D mutants. The mean V1/2 of the triple D mutant (–7 ± 2 mV) was significantly different (**P < 0.01, 1-way ANOVA and post hoc Bonferroni) from WT (–24 ± 3 mV) and the triple A mutant (–26 ± 3 mV). G, conductance; Gmax, maximum G.

 

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  		Table 1. Site-directed mutants

 
To investigate the contribution of each individual phosphorylation site to the regulation of the transient outward K+ current, we investigated the functional consequences of single aspartate mutants to mimic ERK phosphorylation at each individual phosphorylation site. We first investigated the effects of mutation of S616 to an aspartate.

Mutation of S616 to aspartate does not mimic the effect seen in neurons. Surprisingly, mutation of the S616 site to aspartate caused an effect contrary to the effect of the triple D mutant. Mutation of S616D shifted the activation voltage to more hyperpolarized voltages compared with WT (Fig. 3B). The activation curve of the S616D mutant was shifted 11 mV to the left toward more hyperpolarized membrane potentials. The mean V1/2 for the S616D mutant was –35 ± 2 mV compared with WT V1/2 of –24 ± 3 mV. Interestingly, the effect of the S616D mutant is opposite that of ERK on A-type currents in hippocampal dendrites. This finding suggests that phosphorylation at this site does not mediate the effects of ERK on A-type currents in the dendrites of hippocampal pyramidal neurons. The data regarding the 616D mutant suggest that phosphorylation at T602 and T607, the remaining two sites, might cause an effect similar to that seen in response to ERK activation in the dendritic A-type current in intact cells and similar to the triple D site mutants as well. We therefore determined whether mutation of both T602 and T607 to D caused channel inhibition as we anticipated based on neuronal recordings of A-type currents and the effect of the triple D mutant in the oocyte expression system.


Figure 3
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  		Fig. 3. Mutation of T607D mimics the effect of the triple D mutation. A: example of outward K+ current recorded the same day from oocytes expressing the T607D mutant (gray) and WT (black) channels. The maximal outward current from the T607D mutant is scaled up to show the change in activation at lower membrane potentials. B: activation curve showing the normalized conductance vs. test voltage of the WT, T607D, T602D,T607D, and S616D mutants. The activation curves of the T607D and T602D,T607D mutants are shifted to the right, whereas the curve of the S616D mutant is shifted to the left. The slopes of the activation curves were not significantly different: WT 18 ± 0.7, T602D,T607D 18 ± 1, T607D 16 ± 0.2, and S616D 17 ± 1. C: mean ± SE V1/2 (acquired from a Boltzmann fit of the curve) of the WT (–24 ± 3 mV) and T602D,T607D (DD) (–10 ± 2 mV), T607D (–6 ± 3 mV), T607A (–26 ± 3 mV), S616D (–35 ± 2 mV), and S616A (–25 ± 3 mV) mutants. The mean V1/2 values for T607D and T602D,T607D were significantly different (***P < 0.001, **P < 0.01, respectively; 1-way ANOVA with post hoc Bonferroni test) from WT.

 
T602D,T607D mimics the effect seen in neurons. Consistent with this idea, the T602D,T607D double-mutant channel exhibited a shift in the voltage dependence of activation to more depolarized potentials. The T602D,T607D double mutant showed a 14-mV shift in the voltage dependence of activation (Fig. 3B). The mean V1/2 of the T602D,T607D mutant (–10 ± 2 mV) was significantly different (P < 0.01) from WT (–24 ± 3 mV). The double mutant also displayed a slower recovery from inactivation (19 ± 5 ms; n = 6 recordings from oocytes) that was significantly different from WT (Table 1; P < 0.05). Similar to the triple D mutation, the inactivation curve was fit with a single exponential and we determined that the time constant of inactivation of the T602D,T607D mutant (29 ± 6 ms) was significantly different from WT (57 ± 5 ms; P < 0.01).

The T602D,T607D double-mutant channel was quantitatively similar to the triple D mutant channel, as the values for V1/2 and recovery from inactivation were not significantly different from those for the triple D mutant (P > 0.05). This suggests that the effects of the aspartate mutation at T602 and T607 are responsible for the channel inhibition that we observed in the triple D mutant channel.

Overall our data suggest that there are different effects of phosphorylation of Kv4.2 by ERK at the S616 site vs. the T602 and T607 sites. Mutation of T602 and T607 to an aspartate has an overall effect of less current (shift of activation and slower recovery from inactivation), whereas the effect of the aspartate mutation at S616 has the opposite effect, with insertion of a negative charge causing more current and faster recovery from inactivation. It is important to note that the phosphorylation of T602 and T607 mimics the effect of ERK activation on the dendritic A-type current in intact cells. Moreover, together, our data suggest that if all three sites are phosphorylated (mimicked in the triple D mutant), the inhibitory influence dominates over the potentiating effect. That is, we predict that in the native channels, the T602/607 site effect will predominate over the S616 effect if all the sites are phosphorylated simultaneously.

Mutation of T602 to aspartate has no effect. We next sought to determine the effects of mutation of the 602 and 607 sites individually. Mutation of T602 alone to an aspartate (T602D) or to an alanine (T602A) had no significant effect on Kv4.2 current (data not shown). There was no apparent shift in activation voltage of Kv4.2 current, steady-state inactivation, or recovery from inactivation (Table 1). These data suggest that modification of the 602 site is not sufficient to mimic dual modification of the 602 plus 607 sites.

Mutation of T607 to aspartate mimics the effect in neurons. In contrast, mutation of T607 to aspartate caused a pronounced shift toward depolarized potentials in the voltage dependence of activation of Kv4.2 currents. Thus mimicking phosphorylation of T607 causes the current to activate at more depolarized potentials (Fig. 3, A and B). The mutation of T607 to alanine is an important control that suggests that any mutation of this site does not simply cause a conformational change in the protein structure. The activation curve of the T607D mutant also showed an 18-mV shift (–6 ± 3 mV; n = 12) toward more depolarized membrane potentials that was significantly different compared with WT (–24 ± 3 mV; P < 0.001) and T607A (–26 ± 3 mV; P < 0.01). Similar to what we observed with the triple D and T602D,T607D mutants, mutation of T607 to aspartate significantly (P < 0.01) slowed the rate of recovery from inactivation [20 ± 3 ms (n = 10) vs. WT 10 ± 1 ms (n = 25); Fig. 4 ]. The inactivation curve was fit with a single exponential and determined that the time constant of inactivation of the T607D mutant (28 ± 3 ms) was significantly different (P < 0.01) from WT (57 ± 5 ms).


Figure 4
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  		Fig. 4. T607D mutant recovered from inactivation slower than WT. A: current traces (not leak subtracted; capacitance transients are truncated) recorded from the T607D + KChIP3 mutant WT Kv4.2 + KChIP3 from the same batch of oocytes on the same day. The T607D mutant reaches maximal current slower than WT. B: peak current (I) after each hyperpolarized recovery pulse was measured and normalized to the maximal current (Imax). Mean ± SE normalized data were plotted vs. recovery time. That curve was fitted with a single exponential. The T607D mutant recovered from inactivation with a time constant of 17 ± 3 ms (n = 6). This was significantly different from WT (10 ± 1 ms; n = 26) and T607A (9 ± 1 ms; n = 13). n, no. of recordings from oocytes.

 
Mutation of T607 to an aspartate has an effect on the outward K+ current similar to that of both the triple D mutation and the T602D,T607D double mutation. Specifically, the V1/2 and the recovery from inactivation of the T607D mutant channel were not significantly different from the triple D channel or the double-mutant channel. Importantly, these changes are similar to the effects of ERK activation on the A-type current in hippocampal dendrites. A parsimonious interpretation of our data is that ERK phosphorylation of T607 in vivo is a mechanism by which ERK inhibits A-type currents formed by Kv4.2 in neurons. This is suggested by the shift of the activation voltage of the T607D mutant to more depolarized potentials and the decrease in the rate of recovery from inactivation in the T602D and T607D mutants. Because we also observed a decrease in the rate of recovery from inactivation in the T602D mutant, phosphorylation of this site may contribute to channel inhibition as well.

T607D effect requires KChIP coexpression. We previously reported that the functional effects of PKA phosphorylation of Kv4.2 require coexpression with KChIP3 (33). Because phosphorylation of the T607 site is particularly relevant to the A-type currents formed by Kv4.2, we examined the functional effects of the T607D and T607A mutants expressed in the absence of KChIP3 coexpression. In the absence of coexpression with KChIP3, the V1/2 of the T607 mutants (alanine or aspartate) did not show any significant differences compared with WT Kv4.2 also expressed in the absence of KChIP3 (Table 1). Interestingly, although expression of KChIP3 with the WT and T607A channels causes a leftward shift in the activation voltage, only a small shift of the activation voltage is revealed with the T607D mutation (Fig. 5). In addition, no effect was seen on the rate of recovery from inactivation for either mutation (Table 1). Therefore, it appears that coexpression of KChIP3 is necessary for the functional effects of ERK phosphorylation at the carboxy-terminal ERK phosphorylation site (T607). These data are reminiscent of the necessity of KChIP3 for PKA regulation of Kv4.2 as well and may suggest a conserved functional role for KChIPs in conferring phosphorylation-dependent regulation on Kv4.2 currents.

Because KChIP3 coexpression with Kv4.2 is necessary for the functional effects of ERK phosphorylation, the possibility exists that KChIP coexpression may be necessary for phosphorylation of Kv4.2 by ERK. To consider this possibility, we used our antibody directed at Kv4.2 phosphorylated by ERK to determine whether Kv4.2 phosphorylation by ERK was affected by KChIP3 coexpression (Fig. 6, A and B). COS cells were transfected with Kv4.2 + control (GFP) or Kv4.2 + KChIP3. Because KChIP3 increases expression of Kv4.2, we determined the ratio of recognition of the antibodies of Kv4.2 alone to Kv4.2 + KChIP3. The ratio of total Kv4.2 antibody recognition of Kv4.2 to Kv4.2 + KChIP3 (0.66 ± 0.03; n = 3) was not significantly different (P = 0.94) from the ratio of the phospho-Kv4.2 antibody recognition (0.65 ± 0.09; n = 3). These data suggest that KChIP3 is not necessary for ERK phosphorylation of Kv4.2.


Figure 6
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  		Fig. 6. KChIP3 coexpression does not effect ERK phosphorylation of Kv4.2, and phosphorylation of Kv4.2 does not alter surface expression. A: COS-7 cells were transfected with Kv4.2 alone + control (GFP) or Kv4.2 + KChIP3. Cell membrane preparations were run on an SDS gel, and Western blots were probed with total Kv4.2 (TKv4.2, top) or phospho-Kv4.2 (PKv4.2, bottom). B: ratio of antibody recognition for Kv4.2 expressed alone to Kv4.2 + KChIP3. The ratio of antibody recognition was not different for the total Kv4.2 vs. the phosphorylated Kv4.2 antibody. C: triple D mutant channel or triple A mutant channel construct does not show altered surface expression compared with WT. Top: COS-7 cells were transfected with WT + KChIP3, triple D + KChIP3, or triple A + KChIP3. The surface proteins were isolated by biotinylation with an avidin pull-down assay. The biotinylated proteins (surface pool, S) and total cell homogenates (T) were run on an SDS gel, and Western blots with the total Kv4.2 antibody were performed. The surface protein from each of the mutants was normalized to the total protein expression from that mutant. That amount was then compared as a percentage with the WT surface expression. B: quantification revealed that there was no difference in surface expression between WT (100%) and triple D (137 ± 32%) and triple A (101 ± 32%) mutants (P > 0.05, 1-way ANOVA).

 
One caveat to the interpretation of our results is that there are two other minimal ERK consensus sites (simply a serine or threonine followed by a proline) in the carboxy terminus of Kv4.2 at S516 and T567. Although these sites could potentially be phosphorylated by ERK, we did not observe any phosphorylation of these sites in our initial characterization of ERK phosphorylation of the Kv4.2 carboxy terminus (1). Therefore, it is unlikely that they are phosphorylated in the full-length protein. In an additional control experiment (data not shown), we made a GST-fusion protein construct of the WT carboxy terminus and triple A mutant carboxy terminus (T602A, T607A, S616A) and performed in vitro phosphorylation with activated ERK. As expected, the WT carboxy-terminal fusion protein was phosphorylated by ERK/MAPK. Phosphorylation of the triple A mutant carboxy terminus was greatly reduced (24 ± 5% of control; n = 6; normalized to protein concentration), suggesting that blocking phosphorylation at the T602, T607, and S616 sites greatly reduces ERK phosphorylation of the carboxy terminus. These data thereby serve as an independent confirmation of our previous direct sequencing studies and indicate, using the site-directed mutagenesis approach, that these sites are indeed phosphorylated by ERK/MAPK. The residual amount of phosphorylation suggests that the S516 or T567 sites may be phosphorylated in vitro. However, we also cannot rule out the possibility that mutation of the three sites to alanine may alter protein structure and reveal a previously unphosphorylated site.

Protein expression and surface localization. Finally, to determine whether ERK phosphorylation alters protein expression and/or channel surface localization, we expressed the ERK site mutants in the COS-7 cell expression system. This heterologous expression system is more suitable for these studies because levels of protein expression can be highly variable in oocytes. Moreover, we previously observed (38, 45) phosphorylation-dependent and KChIP-dependent alterations in channel trafficking in COS cells. We found no statistically significant effect on total protein expression of any of the site mutants (Table 1). As COS-7 cells have significant basal ERK activity and Kv4.2 phosphorylation (see Fig. 1), these data suggest that ERK phosphorylation of Kv4.2 is not a major determinant of overall Kv4.2 channel expression in these cells.

We also found no significant effect of phosphorylation of the Kv4.2 channel on surface expression (Fig. 6C). The surface Kv4.2 protein (biotinylated and precipitated with avidin beads) was normalized to total Kv4.2 protein (from cell homogenates). The triple A (101 ± 23% of WT; n = 4) or triple D (137 ± 32% of WT; n = 5) mutants were not significantly different from WT surface expression (n = 5), although there was a trend toward increased surface expression for the triple D mutant. These data suggest that ERK phosphorylation of Kv4.2 has no effect on protein expression or surface localization. This observation is consistent with the effects of ERK activation on dendritic A-type current data, as the effects on the A-type current are more rapid than would be expected for changes in total protein or altered protein trafficking (21, 22, 47, 51). Overall, these observations are consistent with our model that the gating effects described above account for the effects of ERK activation in neurons and that direct phosphorylation of the Kv4.2 pore-forming subunit by ERK causes a decrease in the A-type current of dendrites.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have shown herein that mimicking ERK phosphorylation with aspartate mutants at the T607 site causes a decrease in the Kv4.2-mediated outward K+ current. This effect occurs through an increase in the voltage required to activate the channel and a slower recovery from inactivation compared with WT. Before we began these studies, four relevant observations had been established in the literature: 1) ERK modulates A-type currents in pyramidal neuron dendrites (47, 51); 2) Kv4.2 is a likely candidate molecule encoding dendritic A-type currents (32, 35, 43, 44, 50); 3) ERK is capable of directly phosphorylating the Kv4.2 carboxy-terminal domain in vitro (Ref. 1; see also Fig. 1C); and 4) these candidate ERK sites are capable of being phosphorylated in the intact channel in vivo (Fig. 1C; Ref. 1). The present studies provide a direct bridge from these earlier in vitro studies to functional studies in the intact cell. On the basis of our findings, we hypothesize that ERK modulates the A-type current by direct regulation of the Kv4.2 {alpha}-subunit. Our findings support this hypothesis by demonstrating that mimicking ERK phosphorylation of the Kv4.2 {alpha}-subunit is capable of regulating channel biophysical and functional properties. Specifically, we found that mimicking phosphorylation of T607 significantly decreases the current.

Interestingly, the three ERK phosphorylation sites do not appear to have a universal effect. Specifically, introduction of a negative charge at the S616 site causes an effect opposite to that of the T602 and T607 sites. The S616D mutation causes a leftward shift of the activation curve and a faster recovery from inactivation, whereas the S616A mutation slows recovery from inactivation. This effect of the alanine mutation at S616 has two possible explanations: 1) mutation of S616 causes structural changes that affect the Kv4.2 current, specifically altering inactivation kinetics (see below), or 2) S616, and not T602 or T607, is endogenously phosphorylated in the oocyte, and the S616A mutant blocks this effect. Considering the first possible explanation, a structural change in the protein could have results unrelated to phosphorylation at the S616 residue, thus causing an effect independent of phosphorylation. In the second explanation, endogenous phosphorylation in the oocyte could be the result of phosphorylation by ERK or another MAPK. Indeed, activation of ERK vs. other MAPKs has been shown to have antagonistic effects (reviewed in Refs. 10, 28). Finally, although we did not find an effect on surface expression with our mutants in COS-7 cells, we cannot rule out the possibility that these sites may participate in targeting the Kv4 subunits to subcellular domains within a specific cell type.

Mechanism of effects. The functional changes that occur with direct ERK phosphorylation of Kv4.2 appear to be dependent on its interaction with KChIP. For example, we saw no effect on channel properties with mutation of T607 to an aspartate or to an alanine in the absence of KChIP. In addition, we observed an effect of mimicking ERK phosphorylation on the activation voltage as well as recovery from inactivation, two kinetic characteristics that have been shown to be modified by KChIP interaction (4, 5, 16). This suggests that KChIP coexpression is necessary for functional effects as we (33) and others (24) have previously observed for other phosphorylation sites or other biophysical modulation. We cannot rule out the possibility that interactions with other accessory subunits, such as the dipeptidyl peptidase-like proteins (26, 27) may also play an important role in the regulation of the current by phosphorylation.

Functional implications. The present studies implicate the Kv4.2 voltage-dependent K+ channel as a direct target of ERK. In the context of neuronal development and differentiation as well as disease processes, direct ERK/MAPK phosphorylation of Kv4 channel subunits presents a possible mechanism for the acute regulation of K+ currents by growth factors, as has been previously reported (12, 49).

In addition, we and others recently have implicated MAPKs in long-term neuronal plasticity and memory formation and have hypothesized that MAPKs regulate voltage-dependent ion channels in neurons as part of a coordinated mechanism for triggering lasting change in neurons. Previous studies have shown that the dendritic A-type current is modulated by ERK activation (21, 22, 47, 51). Moreover, in an animal model of temporal lobe epilepsy, dendritic A-type currents were reduced along with Kv4.2 protein as well as mRNA. Interestingly, an increase in ERK phosphorylation of the remaining Kv4.2 channels was observed, and kinase inhibition partially reversed the hyperexcitable effects of decreased A-type current (7), suggesting dual regulation of Kv4.2 at a transcriptional as well as posttranslational level. Our analysis shows that the gating properties of Kv4.2 can be modulated dynamically by direct ERK phosphorylation. This modification of Kv4.2 by ERK/MAPK decreases A-type current amplitude and increases the amplitude of back-propagating action potentials in neuronal dendrites. These changes may allow greater depolarization at the synapse and increase the likelihood for the induction of plasticity or, in the case of temporal lobe epilepsy, hyperexcitability. The present studies specifically extend this model by suggesting that the effects of ERK on Kv4.2 function are direct, involving ERK phosphorylation of T607 in the carboxy-terminal cytoplasmic domain of Kv4.2.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by National Institute of Mental Health Grant MH-064620 (to L. A. Schrader) and National Institute of Neurological Disorders and Stroke Grants NS-37444 (to J. D. Sweatt) and NS-039943 (to A. E. Anderson).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Schrader, Dept. of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane Univ., New Orleans, LA 70118 (e-mail: schrader{at}tulane.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1368 - R1375.
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S. Missan, P. Linsdell, and T. F. McDonald
Tyrosine kinase and phosphatase