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The Journal of Neuroscience, December 1, 2002, 22(23):10123-10133
PKA Modulation of Kv4.2-Encoded A-Type Potassium Channels
Requires Formation of a Supramolecular Complex
Laura A.
Schrader1,
Anne E.
Anderson1, 2,
Amber
Mayne1,
Paul J.
Pfaffinger1, and
John David
Sweatt1
1 Division of Neuroscience and
2 Departments of Pediatrics and Neurology, Baylor College
of Medicine, Houston, Texas 77030
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ABSTRACT |
A-type channels, encoded by the pore-forming 
-subunits of the
Kv4.x family, are particularly important in regulating membrane excitability in the CNS and the heart. Given the key role of modulation of A currents by kinases, we sought to investigate the protein structure-function relationships underlying the regulation of these
currents by PKA. We have previously shown the existence of two PKA
phosphorylation sites in the Kv4.2 sequence; therefore, we focused this
study on the Kv4.2 primary subunit. In the present studies we made the
surprising finding that PKA phosphorylation of the Kv4.2 -subunit is
necessary but not sufficient for channel modulation; channel modulation
by PKA required the presence of an ancillary subunit, the
K+ channel interacting protein (KChIP3). Therefore,
these findings indicate a surprising complexity to kinase regulation of
A currents, in that an interaction of two separate molecular events,
-subunit phosphorylation and the association of an ancillary subunit
(KChIP3), are necessary for phosphorylation-dependent regulation of
Kv4.2-encoded A channels by PKA. Overall, our studies indicate that PKA
must of necessity act on a supramolecular complex of pore-forming
-subunits plus ancillary subunits to alter channel properties.
Key words:
KChIP; phosphorylation; neuromodulation; Kv4.2; shal-type; PKA; heart; neuron
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INTRODUCTION |
Potassium currents, specifically
A-type K+ currents, are known to regulate
neuronal membrane excitability. The transient A-type K+ current is present in high densities in
the dendrites of CA1 pyramidal neurons (Hoffman et al., 1997
), where
the neurons receive synaptic input. Rapid activation of these channels
can limit the peak of back-propagating action potentials as well as
modulate incoming synaptic information, exerting profound effects on
hippocampal network communication (Hoffman et al., 1997). These A-type
K+ currents are modulated by PKA
activation (Hoffman and Johnston, 1998), and this modulation of
A-current amplitude regulates the peak of back-propagating action
potentials (Hoffman and Johnston, 1999).
The molecular components that make up this current are unknown;
however, the shal-type channel-forming -subunits
(Kv4.1-Kv4.3) participate in transient A-type current characterized in
neurons throughout the CNS. Specifically, Kv4.2 is a rapidly
inactivating, voltage-gated K+ channel
that activates at membrane potentials above 
40 mV and is sensitive to
4-aminopyridine (Baldwin et al., 1991; Blair et al., 1991; Serodio et
al., 1994, 1996). It is highly likely that Kv4.2 is a pore-forming
subunit of A-type K+ channels in CA1
pyramidal-cell dendrites, because Kv4.2 is highly expressed in
hippocampal pyramidal neuron soma and dendrites (Sheng et al., 1992;
Maletic-Savatic et al., 1995; Tsaur et al., 1997); the pharmacological
and kinetic properties of Kv4.2 expressed in oocytes are similar to the
transient outward currents in dendrites (Blair et al., 1991; Serodio et
al., 1994, 1996; Hoffman et al., 1997). In addition, Kv4.2 most likely
plays a role in transient outward currents in other brain areas
(Serodio and Rudy, 1998; Shibata et al., 1999), including the striatum
and the basal forebrain (Song et al., 1998; Tkatch et al., 2000). The
precise target of kinase modulation of hippocampal transient currents
is not clear, although in previous studies we have shown that the
cytoplasmic domains of Kv4.2 are phosphorylated by PKA (Anderson et
al., 2002). In these previous studies we identified two PKA
phosphorylation sites: Threonine 38 (T38) and Serine 552 (S552)
in the cytoplasmic domains of Kv4.2 (Anderson et al., 2000). Given the
presence of these two phosphorylation sites, and based on previous
results in studies of other channels, a parsimonious hypothesis is that PKA regulation of Kv4.2 is mediated by direct phosphorylation of the
-subunit.
Voltage-dependent K+ channels are
tetramers composed of homodimers or heteromers (within a family; i.e.,
shal) (Jan and Jan, 1992; Pongs, 1992; Chandy and Gutman, 1995). The
Kv4-channel complex contains primary, pore-forming -subunits but can
also contain various 
-subunits or interacting subunits. Auxiliary
subunits are known to interact with channels containing principal
subunits and contribute to the regulation of biophysical properties and the expression levels of K+ channels. One
family of Kv4 interacting proteins, the K+
channel interacting proteins (KChIPs), has been described recently (An
et al., 2000). KChIPs act as chaperones for Kv4.2 and modulate the
kinetic properties of Kv4 channels. Four subtypes of KChIPs (KChIP1,
KChIP2, KChIP3, and KChIP4) have been described and are known to
interact with the N-terminal of Kv4.2 or Kv4.3 (An et al., 2000;
Morohashi et al., 2002). Interestingly, KChIP3 was first cloned as
calsenilin, which interacts with presenilins (Buxbaum et al., 1998) and
has also been described as donnstream regulatory element antagonist
modulator (DREAM), a transcriptional repressor of the prodynorphin gene
(Carrion et al., 1999). KChIP2 and KChIP3 are localized to the
hippocampus (An et al., 2000); thus, the KChIPs are possible candidates
for modulators of Kv4.2 in the hippocampus. In addition, KChIPs are
also Ca2+ binding proteins, which have the
potential for a role in activity-dependent plasticity.
In the present studies, we found that PKA regulation of Kv4.2-encoded
currents required the presence of a KChIP subunit. Despite the ability
of PKA to phosphorylate the Kv4.2 -subunit in the absence of KChIP3,
PKA was unable to alter channel biophysical properties by this
mechanism alone. Thus, regulation of Kv4.2 by PKA appears to require
that PKA act on a complex of pore-forming -subunit plus an ancillary
subunit. This reveals an unexpected complexity to the
structure-function relationships for kinase regulation of membrane
potassium channels.
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MATERIALS AND METHODS |
Functional expression in Xenopusoocytes.
Oocytes were harvested from the ovarian lobe of female
Xenopus laevis frogs. Briefly, the frog was
anesthetized by submersion in 0.15% tricaine. The ovarian lobe was
then surgically removed and placed in
Ca2+-free solution (in
mM: 82.5 NaCl, 2.5 KCl, 1 MgCl2, and 5 HEPES). The frog was allowed to
recover and placed back in the tank. The oocytes were digested in
1.6-2.4 mg/ml collagenase (Roche Diagnostics, Indianapolis, IN)
for several hours. Digested oocytes were then incubated in ND-96, a
solution containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4, supplemented with pyruvic acid (2.5 mM) and gentamicin sulfate (50 mg/ml; Invitrogen, Grand Island, NY). After ~24 hr, oocytes were injected with 30-50 ng
of DNA using a Nanoject microinjector (Drummond Scientific Co.,
Broomall, PA) into the nucleus of stage V to stage VI oocytes. Currents
were recorded after 2-3 d under two-electrode voltage-clamp using an
Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) at room
temperature. Microelectrodes were pulled from filamented glass
(1.5 × 0.86 mm; A-M Systems, Carlsborg, WA) filled with 3 M KCl. The current electrode had a resistance of
0.30-0.50 M
, whereas the resistance of the voltage electrode ranged
from 0.3 to 1.0 M. Currents were leak-subtracted online using P/4
leak subtraction. Data were digitized at 2 kHz and stored using a
Digidata 1200 (Axon Instruments). Current protocols used to obtain data include activation (hyperpolarization to 110 mV then depolarization to +40 mV for 400-800 msec and repeated in 5 mV intervals),
inactivation (depolarization to 0 mV then hyperpolarization to 110 mV
for 650 msec, changing this step by +5 mV intervals), and then
depolarization to 0 mV. For recovery from inactivation for oocytes
expressing Kv4.2 alone, the membrane was hyperpolarized to 110 mV
from 20 mV for 5 msec (with subsequently longer hyperpolarizations
increasing in increments of 50 msec), for a final hyperpolarization of
705 msec, then depolarized to 20 mV. Kv4.2 plus KChIP3 recovered from inactivation more quickly than Kv4.2 alone, so the initial
hyperpolarization pulse was 2 msec, increasing in increments of 10 msec, for a final duration of hyperpolarization of 192 msec.
The chamber was continuously perfused with ND-96 with NaOH at a rate of
3-6 ml/min. Solution changes were achieved by a two-way valve system
(General Valve, Fairfield, NJ). Forskolin (Sigma, St. Louis, MO) was
dissolved in DMSO, stored in 50 mM aliquots at
20°C, and diluted to 50 µM when used. 8-Bromo
(8-Br)-cAMP (Calbiochem, La Jolla, CA) was dissolved in DMSO at a
concentration of 100 mM and then diluted to 100 µM when used. H-89 (Calbiochem) was dissolved in DMSO at
a concentration of 10 mM and used at 10 µM.
As a control, the effect of DMSO (0.1%) alone was tested on
Kv4.2-expressing oocytes. This percentage of DMSO had no effect on
Kv4.2 currents (n = 3).
Data were analyzed using Clampfit (Axon Instruments), Origin (Microcal
Software Inc., Northampton, MA), and Prism (GraphPad Software, San
Diego, CA) programs. Peak currents were obtained and conductance was
determined using a reversal potential of 95 mV. Activation and
inactivation curves were fitted with a Boltzman sigmoidal curve with
the following equation: y = bottom + (top bottom)/[1 + exp(V1/2 x)/slope]). Inactivation time constants were fit in Clampex
(Axon Instruments) with the Simplex method. Only currents that
could be fit with a single exponential in control ND-96 were used and
described here.
DNA preparation and site-directed mutagenesis. The original
Kv4.2 and KChIP cDNA was provided by P. J. Pfaffinger. Both
constructs are in a cytomegalovirus vector. Point mutations were
made using the site-directed mutagenesis kit (Stratagene, La Jolla,
CA). The primers used include 5'-GAGAGAAAAAGGGCCCAGGACGCTCTAATTGTG-3' for the T38 site and 5'-GGAAGTCATAGAGGAGCCGTGCAAG- AACTC-3' for the
S552 site. The double-mutant was made by using the T38 primer on the
S552A DNA. The KChIP3 double mutation was made using the primer
5'-GTGACAAAGGCGGCGGACGGCGCTCTT- CTG-3'. Mutations were confirmed by
restriction enzyme digestion and DNA sequencing. In most cases, the
entire Kv4.2 or KChIP3 region was sequenced to determine whether any
other mutations existed.
Protein expression and purification. The KChIP3 protein was
expressed in Escherichiacoli as glutathione
S-transferase (GST) fusion proteins using methods modified
from Hakes and Dixon (1992). Plasmids containing the KChIP3 cDNAs were
constructed using the GST-fusion vector pGEX-KN (Hakes and Dixon,
1992). 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
(A600) the culture was centrifuged (1000 × g, 15 min, 4°C) (Beckman J2-21M; Beckman Instruments,
Fullerton, CA). The cell pellet was resuspended in 500 ml of LB with
carbenicillin. The bacteria was induced by incubation at room
temperature with 200 µM
isopropyl--D-thiogalactopyranoside for
4 hr 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 -mercaptoethanol and 100 µg/ml
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 (Roche Diagnostics) and 10 mM
MgCl2. The lysate was then centrifuged (1000 × g, 15 min, 4°C; Sorvall RT 6000B; Kenoro Laboratory
Products, Newton, CT) and adjusted to a 2% Triton X-100 concentration.
The GST-fusion protein was purified using glutathione affinity
absorption. Glutathione agarose beads were washed, resuspended in Tris
buffer 1, and then incubated with the lysate for 1 hr at 4°C. The
beads were washed three times with Tris buffer 1 by repeat
centrifugation (100 × g, 5 min, 4°C; Sorvall). 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).
Phosphorylation of KChIP3. KChIP GST-fusion proteins were
incubated at 37°C in reaction mixtures (25 µl) containing 70 ng of
the catalytic subunit of PKA, Tris buffer 2, and ATP mix 1 (100 µM ATP, 100 mM
MgCl2, and 10 µCi
[
-32P]ATP). Reactions were stopped by
boiling for 5 min with sample buffer (30 mM
Tris-HCl, pH 6.8, 200 mM DTT, 40% glycerol, 8%
SDS, and 0.04 mg/ml bromophenol blue). The GST-fusion proteins were separated by SDS-PAGE (10%) and visualized with Coomassie blue staining. Phosphopeptides were identified by autoradiography. As a
control, parallel reactions are performed for GST with and without the
kinases and ATP mix 1. Kinase phosphorylation was also performed.
Phosphopeptide mapping. Phosphorylation reactions were
performed as described above, with some modifications; a preparative scale (reaction volume, 300-400 µl) of the reaction was made. The incubation period was adjusted, based on the time course of kinase
phosphorylation of the fusion proteins. The phosphorylated GST-fusion
protein was separated by SDS-PAGE (10%). After Coomassie blue staining
of the gels, the bands corresponding to the KChIP fusion proteins
were excised, and an in-gel digestion with trypsin was performed
as described previously (Frangioni and Neel, 1993), with minor
modifications. After extraction from the gel, the peptides were
separated using reverse-phase HPLC with absorption monitoring at 214, 254, and 280 nm. Counts per minute in each HPLC fraction were
measured as Cherenkov radiation. Phosphopeptides identified as HPLC
fractions containing high radioactivity were applied to Sequelon
arylamine membranes (Millipore, Bedford, MA) essentially as described
by the manufacturer. After drying, the membrane was rinsed two times
sequentially with 10 ml of methanol, then with water, and finally with
5 ml 10% trifluoroacetic acid and 50% acetonitrile in water.
The membrane was air-dried, cut into pieces with a scalpel, inserted in
a BLOTT cartridge, and sequenced in an Applied Biosystems Model
477A Protein Sequencer with an inline 120 A phenylthiohydantoin (PTH)
Analyzer (Applied Biosystems, Foster City, CA) using optimized cycles.
Instead of butyl chloride, 90% methanol-containing phosphoric acid (15 µl/100 ml) was used to extract the cleaved amino acids. After
conversion, 50% of the sample was transferred to the HPLC for
PTH-amino acid identification; the other 50% was collected in the
instrument fraction collector for the determination of radioactivity by
scintillation counting.
Expression in COS-7cells and Western
blotting. The FuGene 6 Transfection Reagent (Roche Diagnostics)
was used for COS-7 cell transfections with plasmid DNAs of Kv4.2 and/or
KCHIP3 (1:1 ratio, 0.1-2.0 µg/µl). Transfected cells were grown on
35 mm plates to a 2 × 105 cell
density. The cells were then harvested and centrifuged. The cell pellet
was resuspended in 10% SDS with 100 mM DTT, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µM phenylmethylsulfonyl fluoride. Sample buffer
was then added, and the samples were loaded on an SDS-PAGE gel (10%)
for Western blotting. Two antibodies were used for the Western blots,
as follows: (1) the phosphoselective C-terminal antibody (Anderson et
al., 2000) and (2) the general C-terminal antibody (not
phosphoselective). Immunoreactivity was measured using densitometry
(NIH Image). Densitometry data were analyzed with a paired Student's
t test.
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RESULTS |
PKA is a potential critical regulator of Kv4.2-encoded channels in
the CNS and the heart. We had previously identified two sites in the
Kv4.2 protein cytoplasmic domains (amino acids T38 and S552) that are
phosphorylated by PKA. To test the hypothesis that direct
phosphorylation of Kv4.2 by PKA causes a functional modulation of
current, we expressed the primary subunit of Kv4.2 alone in
Xenopus oocytes and manipulated PKA activity in the cell. Forskolin (50 µM) was bath-applied to activate
PKA. Bath application of forskolin (15 min) had no significant effect
on the activation kinetics of Kv4.2 alone (n = 7), as
measured by the V1/2 of control (9 ± 2 mV) and V1/2 in
forskolin (6 ± 2 mV) (p = 0.13) and
slope (20 ± 1 and 21 ± 1) (p = 0.15), respectively (Fig. 1).
Similarly, bath application of 8-Br-cAMP (n = 6; data
not shown) had no significant effect on Kv4.2 when expressed alone in
oocytes. The V1/2 of the control was
10 ± 2 versus 8 ± 2 (p = 0.12)
in 8-Br-cAMP; the slope was 20 ± 2 in the control and
20 ± 2 in 8-Br-cAMP (p = 0.92). In
addition, there was no effect on steady-state inactivation or on the
time of recovery from inactivation. Indeed, this concentration of
forskolin has been shown to activate PKA in oocytes
(Schorderet-Slatkine and Baulieu, 1982), and we have shown that PKA is
capable of phosphorylating the Kv4.2 -subunit at both the C- and
N-terminals of Kv4.2 in COS cells (see Fig. 8) and hippocampal slices
using phosphospecific antibodies (Anderson et al., 2000). Therefore, to
our surprise, when Kv4.2 was expressed in Xenopus oocytes,
the activation of PKA with forskolin or cAMP analog had no significant
effect on the kinetics of the K+
current.
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Figure 1.
PKA activation does not modulate transient outward
current when Kv4.2 is expressed alone. A, Activation
curve of Kv4.2 in the control (squares) and in forskolin
(50 µM; triangles). B, Mean
current-voltage plot of the peak amplitude of the current versus test
voltage for the control and forskolin for all cells tested
(n = 7). C, Bar graph of the mean
V1/2 of each cell fitted individually
(Boltzman sigmoidal). The V1/2 in control
was 9 ± 1.7 mV, which did not differ significantly from the
5.7 ± 2.3 mV in forskolin. D, Raw current trace
of depolarization to +20 mV in control (black) and
forskolin (gray) (forskolin current is
scaled up to the amplitude of the control current because forskolin
caused a small decrease in amplitude). E, Scatterplot of
the time constant of inactivation of the current evoked with a
depolarization to +20 mV fitted with a single exponential (from peak to
230 msec) in the control (triangles) and in forskolin
(diamonds). F, Raw current trace showing
the current in forskolin scaled up to the control amplitude to
illustrate the inactivation time constant further. Forskolin does not
significantly alter the time constant of inactivation of Kv4.2
expressed alone.
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Coexpression of Kv4.2 and KChIP3
The KChIPs are a family of interacting proteins that have been
shown recently to interact with the Kv4 family of primary subunits (An
et al., 2000). Interaction of the KChIPs and Kv4.2 causes various
changes in the kinetics of Kv4.2. It has been shown previously that
coexpression of the KChIPs with Kv4.2 caused an increase in current
density, an increase in the rate of recovery from inactivation, an
increase in the time constant of inactivation, and a shift of
activation and inactivation curves in Chinese hamster ovary cells and oocytes (An et al., 2000; Decher et al., 2001; Beck et al.,
2002). Might KChIP be an ancillary subunit necessary for PKA modulation
of Kv4.2? Given our surprising finding described above indicating a
lack of PKA regulation of Kv4.2 -subunit alone, we set out to test
this hypothesis. As a first step, we assessed the effect of KChIP3 on
Kv4.2 channel biophysical properties in our experimental system (Fig.
2). In our preparation, coexpression of
KChIP3 with Kv4.2 shifted the V1/2
activation toward the left 11 mV in the hyperpolarizing direction
(23 ± 1.0 mV; slope, 16 ± 0.3; n = 13)
compared with Kv4.2 alone (12 ± 1 mV; slope, 19 ± 1;
n = 11), and shifted inactivation +15 mV (63 ± 1 mV; slope, 4 ± 0.1 vs 78 ± 1 mV; slope, 6 ± 0.2) and strikingly increased the rate of recovery from inactivation at
110 mV (
= 70 ± 3 msec for Kv4.2 alone and 12 ± 1 msec for Kv4.2 plus KChIP3) (Fig. 2A-D). The time
constant of inactivation, fitted with one exponential, was increased
from 20 ± 1 msec in Kv4.2 alone and 49 ± 3 msec in Kv4.2
plus KChIP3. Overall, these altered properties attributable to
coexpression with KChIP3 made the properties of the K+
current more similar to native A currents observed in neurons and
cardiac myocytes.
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Figure 2.
Modulation of Kv4.2 current by KChIP3.
A, Representative example of transient outward current
recorded from an oocyte expressing only Kv4.2. B,
Example of transient outward current recorded from an oocyte when
KChIP3 is coexpressed with Kv4.2. C, Steady-state
activation and inactivation curves for Kv4.2 alone
(squares) and Kv4.2 plus KChIP3
(triangles). D, Plot of the time of
recovery from inactivation for Kv4.2 expressed alone
(squares) and with KChIP3 (triangles).
KChIP3 speeds the recovery from inactivation (see Table 1).
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Moreover, KChIP3 coexpression rescued the PKA regulation of Kv4.2. In
contrast to what was observed with Kv4.2 alone, activation of PKA with
forskolin caused a significant shift in the activation curve of Kv4.2
coexpressed with KChIP3 (Fig.
3A). The activation curve of
Kv4.2 with KChIP3 was shifted to the right by 6 mV toward more
depolarized membrane potentials from 23 ± 1 to 17 ± 2 mV (n = 13; p = 0.005) (Fig. 3). The
slope was not significantly affected: 16 ± 0.3 compared with
17 ± 0.6 in forskolin (p = 0.3). The
amplitude of the current decreased by 17 ± 2% compared with the
control amplitude in the presence of forskolin (at +20 mV) (Fig.
3D). This effect of forskolin was reversible (Fig.
3B). No significant effect was seen on steady-state
inactivation (the V1/2 of inactivation
was 62 ± 2 mV in control and 63 ± 2 mV in forskolin) or
on recovery from inactivation (Fig. 3D).
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Figure 3.
KChIP3 coexpression is required for modulation of
the current by PKA. A, Activation curve of Kv4.2 plus
KChIP3 current in the control (squares) and forskolin
(triangles). B, The current evoked from
depolarizing pulses to +20, 5, 30, and 60 mV in the control
(black, left), 15 min after the start of
forskolin application (gray,
middle), and 15 min after washout (black,
right). C, Mean current-voltage plot of
the peak amplitude of the current versus test voltage for the control
and forskolin for all cells tested (n = 13).
D, Time of recovery from inactivation for Kv4.2 plus
KChIP3 in control and in forskolin. Forskolin does not significantly
alter the recovery from inactivation.
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Coexpression with KChIP3 also revealed an effect of forskolin on
channel-inactivation properties (Fig. 4).
The inactivation rate (at +20 mV) was best fitted with a single
exponential in a control solution in 10 of 13 oocytes (50 ± 3 msec). However, after forskolin application, a double exponential was
necessary to fit the inactivation rate in 7 of 10 oocytes, with a fast
time constant of 13 ± 3 msec. The slow time constant increased to
74 ± 7 msec (Fig. 4). An increase in channel inactivation rate is a possible mechanism for the decreased amplitude and change in the
number of channels open at certain potentials. Regardless, these observations are consistent with an interaction of KChIP3 and PKA
phosphorylation (specifically, that KChIP3 is necessary for PKA
regulation of Kv4.2).
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Figure 4.
Forskolin modulates the inactivation kinetics of
Kv4.2 plus KChIP3. A, The time constant of inactivation
is fitted with a single exponential in the control and a double
exponential in forskolin. In a representative example of the
current evoked by a depolarization to +20 mV, the forskolin current is
scaled up to the control amplitude to compare the decay phases. Only
currents that could be fitted by a single exponential in the control
were evaluated. B, Scatterplot showing the single
exponential in the control (squares) and the fast and
slow time constant in forskolin (fsk)
(triangles).
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The control analog for forskolin, 1,9-dideoxyforskolin (50 µM) was also tested (Fig.
5A). Dideoxyforskolin had no
effect on the activation curve and
V1/2 (p = 0.1;
n = 3). A reduction of the time constant of
inactivation (65 ± 5 to 45 ± 2 msec) was observed, but
dideoxyforskolin did not cause the current to be fitted by a double
exponential (Fig. 5A, inset).
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Figure 5.
The effect of forskolin is not mimicked by
1,9-dideoxyforskolin and is mimicked by 8-Br-cAMP and blocked by H-89,
the PKA inhibitor. A, Activation curve of Kv4.2 plus
KChIP3 in the presence of 1,9-dideoxyforskolin (Ddfsk;
50 µM). Dideoxyforskolin did not cause a significant
shift in the activation curve (inset): Example of
currents evoked by a depolarizing pulse to +20 mV in control
(black) and dideoxyforskolin
(gray) showing the effect of dideoxyforskolin on
the current. The dideoxyforskolin current is scaled up to the size of
the control. Dideoxyforskolin did not cause the inactivation time
constant to be fitted by two exponentials, but it did have a small
effect on amplitude and on the time constant of inactivation.
B, Activation curve of Kv4.2 plus KChIP3 treated with
8-Br-cAMP (100 µM). C, The effect of
forskolin on the activation curve is blocked by H-89 (10 µM). D, Raw current traces showing the
effect of forskolin in H-89. H-89 (10 µM) blocked
forskolin-induced changes in the time constant.
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The forskolin-induced shift of the activation curve is mimicked by
8-Br-cAMP (100 µM) (Fig. 5B). 8-Br-cAMP caused
a rightward shift of the activation curve (6 mV) from 23 ± 1 to
17 ± 1 mV (n = 5; p = 0.003),
and application of 8-Br-cAMP decreased the peak amplitude of the
current at +20 mV by 14 ± 4%. The slope of the activation curve
was not significantly affected (15 ± 0.3 vs 15 ± 0.4).
These observations strongly support the interpretation that the effects
of forskolin are attributable to the elevation of the cAMP levels.
The effect of forskolin was blocked by the PKA inhibitor H-89 (10 µM) (Fig. 5C,D). H-89 alone caused a small
(not significant) shift in V1/2
(25 ± 2 mV in the control vs 23 ± 1 mV in H-89; n = 4) (Fig. 5D). In the presence of H-89
(with a 10 min preincubation), there was no significant
forskolin-induced shift in the voltage dependence of activation
(23 ± 1 mV in H-89 vs 24 ± 1 mV in forskolin;
p = 0.55). In addition, the slopes of the activation curves were not significantly different. H-89 also blocked the effect
of forskolin on the time constant of inactivation (at +20 mV,
49.3 ± 9 msec in H-89 vs 49 ± 6 msec in H-89 plus
forskolin) (Fig. 5F). Thus, we found that the effects
of forskolin were mimicked by 8-Br-cAMP and blocked by H-89, a specific
PKA inhibitor, strongly indicating that these effects are indeed
attributable to PKA activation. These findings suggest that
phosphorylation of some component of the Kv4.2/KChIP protein complex is
necessary for modulation of the channel properties.
Mutation of phosphorylation sites
The effects of phosphorylation on the Kv4.2/KChIP complex could be
attributable to phosphorylation of the -subunit, phosphorylation of
KChIP, or both. As mentioned, we have shown previously that the N- and
C-terminal cytoplasmic domains of Kv4.2 are phosphorylated at T38 and
S552, respectively (Anderson et al., 2000). Therefore, we first tested
whether phosphorylation of Kv4.2 at these sites is necessary for PKA
modulation of Kv4.2/KChIP. Thus, we mutated T38 and S552 to an alanine
to block phosphorylation at these sites (see Materials and Methods).
However, when expressed without KChIP3, each mutant had kinetic
characteristics similar to the wild type expressed alone (Table
1), and the time constant of inactivation was modestly but significantly decreased in the N-terminal mutant and
increased in the C-terminal mutant (Table 1). In addition, the
mutations did not significantly affect the interaction of Kv4.2
with KChIP3, because the kinetics (including increased current density
and recovery from inactivation) was similar to wild type plus
KChIP3 (Table 1). These data indicate that the mutation of T38 or S552
to alanine does not grossly alter the biophysical properties of the
channels or the interaction with KChIP3.
T38A
Each mutant was then coexpressed with KChIP3. When the T38A mutant
was coexpressed with KChIP3, forskolin caused a shift in the activation
curve similar to that seen with wild type (Fig. 6). The control
V1/2 was 23 ± 2 mV compared
with 16 ± 1.5 mV in forskolin (p < 0.05; n = 7); there was no change in the slope of the
activation curve (17.5 ± 0.85 in the control vs 17.4 ± 0.2 in forskolin). The amplitude was decreased by 28 ± 5% (at 20 mV)
in the presence of forskolin (Fig. 6B).
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Figure 6.
Phosphorylation of the PKA N-terminal
site is not required for modulation of the current by PKA. The
N-terminal PKA phosphorylation site (T38) was mutated to an alanine.
A, Activation curve of T38A plus KChIP3 current in the
control (squares) and forskolin
(triangles). B, Mean current-voltage
plot of the peak amplitude of the current versus the test voltage for
the control and forskolin for all cells tested (n = 7). C, A representative example of the current evoked by
depolarizing pulses in control (black) and forskolin
(gray). D, top,
Example of current evoked by a depolarization to +20 mV in control
(black) and forskolin (gray).
Forskolin current is scaled up to the control amplitude to compare the
decay phases. Forskolin caused an increase in the rate of inactivation.
D, bottom, Scatterplot showing that the
time constant of inactivation is fitted with a single exponential in
the control and a double exponential in forskolin (Fsk).
Only currents that could be fitted by a single exponential in the
control were evaluated. Currents from five cells were fitted with a
single exponential in control. For all five, the decay was fitted
with a double exponential in forskolin. E, Time of
recovery from inactivation for Kv4.2 plus KChIP3 in the control and in
forskolin. Forskolin does not significantly alter the recovery from
inactivation.
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The time constant of inactivation in cells expressing the T38A plus
KChIP3 mutant was affected by forskolin in a manner similar to
wild type. The decay of inactivation at +20 mV was fitted with a single
exponential in the control (42 ± 1 msec; n = 5);
in forskolin all cells were fitted with a double exponential as well,
with a fast time constant of 11 ± 1 msec and the slower time
constant increased to 77 ± 5 msec (Fig. 6D).
The recovery from inactivation was not affected by forskolin (Fig.
6E). Overall, these data suggest that PKA
phosphorylation of T38 is not involved in PKA regulation of the
biophysical properties of the Kv4.2/KChIP3 complex.
S552A
In contrast, phosphorylation at S552 in the complex was essential
for channel modulation. Thus, mutation of the S552 site to an alanine
completely blocked forskolin-induced regulation of the properties of
the Kv4.2/KChIP complex. Figure 7 shows
the lack of effect of forskolin on the S552A mutant coexpressed with KChIP3. The V1/2 of S552A plus KChIP3
in the control and forskolin was not significantly different
(p = 0.3). The
V1/2 for control was 20 ± 1 mV
and 19 ± 1 mV in forskolin (n = 8). Also, there was no effect on the slope (19.4 ± 0.5 in the control vs 20 ± 0.3 in forskolin). The amplitude was decreased by only 7 ± 5% in forskolin (at 20 mV), compared with 17 ± 2% in the control and 28 ± 5% in the T38A mutant (Fig. 7B).
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Figure 7.
Phosphorylation of the PKA
C-terminal site is required for modulation of the current by PKA.
A, The C-terminal PKA phosphorylation site (S552) was
mutated to an alanine. The activation curve of S552A plus KChIP3
current in the control (squares) and forskolin
(triangles) is shown. B, Mean
current-voltage plot of the peak amplitude of the current versus test
voltage for control and forskolin for all cells tested
(n = 9). C, The current evoked from
depolarizing pulses in control (black) and forskolin
(gray). Forskolin did not affect the current.
D, top, An example of the current evoked
by a depolarization to +20 mV in the control and in forskolin.
Forskolin did not have an effect on the time constant of inactivation
in the S552A mutant. D, bottom, The time
constant of inactivation of the current from nine cells is fitted with
a single exponential in control. Forskolin (Fsk) forced
a double exponential in only three cells. E, Time of
recovery from inactivation for Kv4.2 plus KChIP3 in the control and in
forskolin. Forskolin does not significantly alter the recovery from
inactivation.
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In addition, the time constant of inactivation in cells expressing
S552A was significantly longer (80 ± 11 msec; p = 0.003) than in wild type (48.7 ± 3.3 msec) or T38A (41.7 ± 1 msec) when coexpressed with KChIP3. After forskolin application, only
three of eight decays at +20 mV were fitted with double exponentials. The fast time constant was 20 ± 3.0 msec (n = 3),
and the long time constant was 102 ± 6 msec (n = 8) (Fig. 7D). These data suggest that phosphorylation of
the S552 site is necessary for the effect of PKA activation on Kv4.2
coexpressed with KChIP3.
We wished to confirm that the mutation of S552 to an alanine did indeed
block PKA phosphorylation of the C-terminal site. Therefore, we
expressed wild-type Kv4.2 and the S552A mutant ± KChIP3 in the
COS cell expression system. Western blots on cells expressing Kv4.2
plus KChIP3 probed with a selective antibody directed against the 552 phosphorylation site revealed immunoreactivity for the C-terminal site
in cells expressing Kv4.2 plus KChIP3 but not in cells expressing S552A
plus KChIP3 (n = 3) (Fig.
8), suggesting that site S552 was not
phosphorylated in the mutant. In our previous studies we observed that
the Kv4.2 subunit when expressed by itself is a substrate for PKA
(Anderson et al., 2000). As a control for the present studies, we
confirmed this observation (Fig. 8B). These data show
that although the Kv4.2 -subunit by itself is phosphorylated at
S552, KChIP3 coexpression is necessary for modulation of the current by
phosphorylation.
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Figure 8.
Mutation of S552A blocks the immunoreactivity for
the C-terminal phosphospecific antibody. A, Western blot
of a COS cell homogenate showing immunoreactivity for the PKA
C-terminal (CT) phosphospecific site
(Phospho-site) (top) and total Kv4.2
(bottom). The phosphospecific antibody recognizes only
Kv4.2 plus KChIP3 and not the S552A mutation. B, COS
cells were transfected with Kv4.2 alone and treated with DMSO or
forskolin (50 µM). Western blots of cellular homogenates
show that forskolin caused phosphorylation of the C-terminal site, as
evidenced by the increase in immunoreactivity for the PKA
C-terminal-specific antibody (top) and no change in
total protein (bottom).
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In an additional series of control experiments, we confirmed that the
double mutation (T38A, S552A) also showed no modulation by forskolin
(data not shown). The V1/2 of
T38AS552A plus KChIP3 in control and forskolin was not significantly
different (n = 9; p = 0.3). The
V1/2 for control was 21 ± 2 and 18 ± 2 mV in forskolin. Also, there was no effect on the
slope (17 ± 0.6 vs 16.1 ± 0.6 in forskolin). The time
constant of inactivation of the double mutant plus KChIP3 was 57 ± 4 msec (n = 7). After forskolin application, only
two of seven decays were better fitted with two exponentials, the fast
time constant was 9.2 ± 2, and the slower time constant was
103 ± 10.6 for all seven cells (data not shown).
KChIP3 phosphorylation
Thus, phosphorylation at S552 appears to be essential for PKA
regulation of the Kv4.2/KChIP complex, although it is clearly not
sufficient to cause modulation of the Kv4.2 -subunit alone. We
wondered whether PKA phosphorylation of KChIP3 might be a missing component necessary for PKA modulation of Kv4.2. One prediction of this
hypothesis is that PKA should phosphorylate KChIP3. We used in
vitro phosphorylation, peptide mapping, and direct amino acid
sequencing and found that indeed KChIP3 is a PKA substrate (Fig.
9). KChIP3 is phosphorylated by PKA at
serine 14 (S14), with a minor site at serine 11 (S11) (Fig. 9).
Although other PKA phosphorylation sites may exist within KChIP3, we
used this information to make site-directed mutants. We mutated the
major site (S14) site to an alanine to determine whether
phosphorylation of this site is necessary for PKA modulation of the
Kv4.2/KChIP complex.
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Figure 9.
KChIP3 is phosphorylated by PKA. A,
KChIP3 protein sequence. Inspection of the KChIP3 sequence reveals
several consensus sites for PKA phosphorylation. KChIP3 was expressed
as a GST-fusion protein in bacteria. The purified GST-fusion protein
was reacted with PKA and -32P in vitro.
B, Reaction products were separated by SDS-PAGE and
stained with Coomassie blue (right). Autoradiography was
performed to analyze the efficacy of KChIP3 as a substrate for PKA.
C, Control experiment showing 32P
incorporation in KChIP3 in the presence of PKA; however, there was no
32P incorporation when PKA was not included in the
experiment. D, The peptide was digested with trypsin,
and two fragments were determined to contain radioactivity. One of the
sequences determined by automated amino acid sequencing corresponded to
amino acids 10-26 in the KChIP3 sequence. S14 was found to be a major
site and S11 a minor site, as indicated by asterisks above
serines.
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Mutation of S14 in KChIP3 to an alanine did have a significant effect
(relative to wild-type KChIP3) on the
V1/2 of Kv4.2 plus KChIP (18 ± 1 vs 23 ± 1 mV for Kv4.2 plus wild-type KChIP; p = 0.003) and recovery from inactivation properties,
but it had no effect on steady-state inactivation when the mutant
KChIP3 was coexpressed with Kv4.2. Mutation of the S14 site in
KChIP3 did not block the effect of PKA activation on the modulation
of the properties of the Kv4.2/KChIP complex (Fig.
10). The
V1/2 activation was 18 ± 1 mV
in control and 14 ± 1 mV in forskolin (n = 10; p < 0.05). The mean time constant of inactivation at
+20 mV was 52.2 ± 2.3 msec. After forskolin application,
inactivation was fitted with a double exponential (12 ± 1 and
81.4 ± 5.5 msec) in all 10 cells.
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Figure 10.
Phosphorylation of the PKA site S14 in the KChIP3
sequence is not required for modulation of the current by PKA.
A, The PKA phosphorylation site (S14) of KChIP3 was
mutated to an alanine. The activation curve of wild type plus S14A
KChIP3 current in control (squares) and forskolin
(triangles) is shown. B, Examples of
current evoked from depolarizing pulses in control
(black) and forskolin (gray).
C, The time constant of inactivation is fitted with a
single exponential in the control and a double exponential in forskolin
(Fsk). The currents from six cells were fitted by a
single exponential in the control. Forskolin caused a double
exponential decay in all six cells. The inset shows an
example of the current; the forskolin current is scaled up to the
control amplitude to compare the decay phases. D, Time
of recovery from inactivation for Kv4.2 plus KChIP3 in the control and
in forskolin. Forskolin does not significantly alter the recovery from
inactivation.
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We sought to determine whether phosphorylation of both S11 and S14
within the KChIP3 sequence is necessary for the modulation of the
current mediated by Kv4.2 (Fig. 11).
Therefore, we mutated both S11 and S14 to alanines. Kv4.2 coexpressed
with S11S14A KChIP3 showed no significant change compared with wild
type on activation (V1/2 = 24 ± 2 mV; n = 9). However, this KChIP3 mutant did show a
significant shift in the time constant of inactivation (68 ± 2.5 msec; p = 0.002). The double KChIP3 mutant still
demonstrated PKA modulation of channel properties. Forskolin
application caused a significant (p < 0.01)
shift in the V1/2 to 20 ± 1.5 mV (n = 9) (Fig. 11). Similar to wild type, forskolin
application forced a double exponential fitted with a fast time
constant of 11 ± 2 msec and a slow time constant of 111 ± 10 msec in all cells tested (n = 9). In both KChIP
mutants, the S14A and S11AS14A, the amplitude was reduced by
forskolin by a larger amount than in the control or the T38A
mutant. The amplitude was reduced by 33 ± 2% in forskolin
relative to control in the S14 mutant; forskolin caused a greater
reduction in amplitude of the current evoked by a depolarization to +20
mV with the S11S14A mutant also, decreasing the amplitude by 39 ± 4%. This amount of current reduction is significantly more than that
seen in the Kv4.2 primary subunit mutants; therefore, additional
investigation is warranted to determine the mechanisms of this effect.
In addition to these effects, forskolin also significantly slowed the
recovery from inactivation. This suggests that the PKA phosphorylation
sites in KChIP3 sites may play a role in maintaining the speed
of recovery from inactivation, but that the overall PKA phosphorylation
of sites S11 and S14 in KChIP3 is not necessary for PKA modulation of
the ion-channel complex.
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Figure 11.
Phosphorylation of PKA sites S14 and
S11 in the KChIP3 sequence is not required for modulation of the
current by PKA. A, The PKA phosphorylation sites (S11
and S14) of KChIP3 were mutated to alanines. The activation curve of
wild type plus S11S14A KChIP3 current in the control
(squares) and forskolin (triangles) is
shown. B, Mean current-voltage plot of the peak
amplitude of the current versus test voltage for the control and
forskolin for all cells tested (n = 9).
C, Examples of the current evoked from depolarizing
pulses in control (black) and forskolin
(gray). D, The time constant of
inactivation is fitted with a single exponential in the control and a
double exponential in forskolin (Fsk). The currents from
nine cells were fitted by a single exponential in the control.
Forskolin caused a double exponential decay in all nine cells. The
inset shows an example of the current; the forskolin
current is scaled up to the control amplitude to compare the decay
phases. E, Time of recovery from inactivation for Kv4.2
plus KChIP3 in the control and in forskolin. Forskolin significantly
alters the recovery from inactivation.
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DISCUSSION |
Our data indicate that two distinct molecular events are necessary
for PKA regulation of the Kv4.2 pore-forming -subunit. First, the
-subunit must be phosphorylated at S552. In addition, for this
phosphorylation event to be efficacious, an ancillary subunit (KChIP3
in our experiments) must also be present. This indicates that the Kv4.2
pore-forming subunit and the KChIP ancillary subunit are operating as a
functional supermolecular complex to allow modulation by
phosphorylation. This indicates a surprising complexity to the protein
structure-function relationships for A-current modulation by PKA.
Direct phosphorylation of the pore-forming -subunit is necessary but
not sufficient for modulation of channel biophysical properties;
additional allosteric effects by an ancillary subunit appear to be necessary.
This complexity of A-current regulation by PKA is further compounded by
the likelihood that PKA phosphorylates additional sites in the complex.
Thus, T38 in Kv4.2 and S11 and S14 in KChIP3 likely can be
phosphorylated by PKA. The roles of these additional phosphorylation
events remain mysterious. However, there are intriguing possibilities.
We have found previously that the T38-phosphorylated form of Kv4.2 is
differentially localized from the S552 variant, suggesting perhaps that
T38 phosphorylation may participate in channel subcellular distribution
(Varga et al., 2000). Other studies have also shown a role for
post-translational modification in the regulation of channel
localization (Ma and Jan, 2002). PKA phosphorylation of KChIP3 also has
interesting potential roles as well, because KChIP3 can function not
only as a Kv4.2 ancillary subunit but also as a calcium sensor and as a
transcriptional regulator. KChIPs have four EF-hand-like
domains and bind calcium ions (An et al., 2000). The function of these
Ca2+ binding domains in the regulation of
Kv4.2 is unknown; however, a Ca2+
dependence of transient K+ currents has
been reported previously (Bourque, 1988; Fisher and Bourque, 1998; Song
et al., 1998). Future studies are necessary to determine whether PKA
phosphorylation of KChIP regulates these functions.
In our studies, we observed that Kv4.2 is modulated by coexpression
with KChIP3. This is similar to what has been reported previously and
makes the Kv4.x-encoded channel behave much more like a native A
current in heterologous expression systems. Kv4.x currents in
expression systems are slightly different from that reported in the
native brain (Rudy et al., 1988; Chabala et al., 1993; Serodio et al.,
1994, 1996), and low-molecular-weight molecules extracted from the
brain can correct this difference when coexpressed with Kv4.x
-subunits (Rudy et al., 1988; Chabala et al., 1993; Serodio et al.,
1994, 1996). Indeed, a recent study shows that KChIP1 coexpression is
necessary to see the effects of arachidonic acid, similar to what is
seen in the native system (Holmqvist et al., 2001). We also found that
coexpression with a KChIP3 ancillary subunit was necessary to
reconstitute an additional native-channel function: modulation by PKA.
How might phosphorylation of the C-terminal lead to changes in the
voltage dependence of activation? Phosphorylation of cytosolic domains
is known to regulate K+-channel function
(Levitan, 1994; Jonas and Kaczmarek, 1996), including effects on
voltage-dependent activation (Murakoshi et al., 1997). Moreover, PKC
phosphorylation of Kv3.4 has been shown to remove fast inactivation,
converting a rapidly inactivating K+
current to a noninactivating one (Covarrubias et al., 1994). We suggest
that phosphorylation actually increases the rate of inactivation.
Indeed, Kv4.1 has been shown to inactivate via interactions of the C
and N terminals (Jerng and Covarrubias, 1997; Jerng et al., 1999).
Therefore, we suggest that an interaction of the N-terminal (through
binding to KChIP) and the C-terminal phosphorylation is involved in PKA
modulation of Kv4.2.
It is interesting to consider the possibility that other Kv4.2
ancillary subunits might be able to substitute for KChIP. In particular, -subunits have been shown to alter channel properties (Rettig et al., 1994; Heinemann et al., 1995; Leicher et al., 1998),
act as chaperone proteins that promote and/or stabilize the
cell-surface expression of -subunits (Levin et al., 1996; Shi et
al., 1996; Nagaya and Papazian, 1997; Trimmer, 1998; E. K. Yang et
al., 2001), or transduce phosphorylation-dependent alterations in
K+ channel properties (Jin et al., 2002),
similar to what we have observed in this study. These subunits lack
putative transmembrane domains and potential glycosylation sites or
leader sequences, suggesting that they are cytoplasmic proteins (Scott
et al., 1994). It will be interesting in the future to determine
whether -subunits also have in common with KChIPs the capacity to
confer PKA modulation.
Kv4.2 is also a substrate for the MAP kinase (MAPK) extracellular
signal-regulated kinase (ERK); ERK regulation of the dendritic transient K+ currents plays an important
role in the neuromodulation of hippocampal pyramidal neurons (Watanabe
et al., 2002; Yuan et al., 2002). It will be interesting to see whether
ERK modulation of Kv4.2 depends on the presence of an ancillary
subunit, as does PKA modulation. Moreover, because both PKA and ERK can
directly phosphorylate the Kv4.2 -subunit, it is intriguing to
consider that there might be functional interactions of these two
molecular events. It has been shown previously that activation of PKA
or PKC caused a shift in the activation curve of transient outward
K+ currents in hippocampal dendrites
(Hoffman and Johnston, 1998). PKA activation has been shown to couple
to ERK activation in the hippocampus (Roberson et al., 1999). This
effect of PKA and PKC activation on the transient outward
K+ current is blocked by the ERK/MAPK
inhibitor U0126 (Yuan, 2002). In addition, glial cell line-derived
neurotrophic factor and 8-Br-cAMP have been shown to increase cell
excitability via reduction of A-type K+
currents in midbrain dopaminergic neurons, an effect that is mediated
by ERK/MAPK (F. Yang et al., 2001). Because U0126 blocked the effect of
PKA activation in the endogenous system, we would predict that
phosphorylation of one (or several) of the previously characterized
ERK/MAPK sites (Adams et al., 2000) may also be necessary for the
PKA-dependent modulation of Kv4.2 or Kv4.3 channels in vivo.
This would be one specific example of the general hypothesis that there
are functional interactions of the various phosphorylation sites
identified on Kv4.2. This complexity of regulation would allow Kv4.2
(plus interacting subunits) to serve as a novel locus for coincidence
detection of various signaling mechanisms.
One interesting issue is whether other Kv4 family ion channels are
similarly regulated as part of a supramolecular complex. Indeed,
dopaminergic neurons express Kv4.3 (Serodio and Rudy, 1998; Liss et
al., 2001), which is highly homologous to Kv4.2. Although the PKA or
ERK phosphorylation sites on Kv4.3 have not been mapped, several
consensus sequences for both kinases exist within the Kv4.3 sequence;
it is highly likely that either kinase can phosphorylate Kv4.3.
Specifically, in the context of the present findings it appears that
the S552 site is conserved in Kv4.3, so similar mechanisms to what we
have observed with Kv4.2 may apply to Kv4.3 as well.
What functional roles might the PKA modulation of Kv4.2 play in the
intact cell? Transient A-type K+ current
exists at high density in hippocampal pyramidal cell dendrites; these
currents play a major role in pyramidal neuron excitability (Hoffman et
al., 1997). Recent work by Quirk et al. (2001) has shown a correlation
between learning behavior and back-propagating spike amplitude, which
is subject to regulation by hippocampal A currents (Hoffman et al.,
1997), thus suggesting that modulation of these currents plays a role
in learning and memory. These A-type currents are modulated by kinase
activation, and this modulation has been shown to regulate the
amplitude of back-propagating action potentials, which can have
dramatic effects on the induction of long-term potentiation and
information processing (Hoffman and Johnston, 1998, 1999). Although the
molecular subunits of these currents are unknown, the primary subunit
protein, Kv4.2, is an excellent candidate, because it exists in the
pyramidal cell dendrites and exhibits properties similar to the native
current. The Kv4 family of K+ channels has
been shown recently to interact with the KChIP family of accessory
subunits in vitro, and they are likely an important component of the K+-channel complex.
Modulation of the properties of these channels (e.g., voltage
dependence, inactivation rate, number of channels, and distribution)
represents a powerful potential site for the regulation of pyramidal
neuron excitability in long-term potentiation. Thus, these
transient outward currents (most likely composed of Kv4.2 -subunits
and other -subunits or interacting proteins) appear to be ideally
suited, both in terms of their biophysical properties and
subcellular localization, for contributing significantly to the
regulation of pyramidal neuron excitability and synaptic responsiveness.
In summary, we have demonstrated a novel mechanism for ion-channel
regulation by kinases. We show that interaction of the Kv4.2
-subunit with an interacting subunit (KChIP3) is necessary for
modulation by PKA phosphorylation. These data indicate a surprising complexity to the structure-function relationships for the regulation of ion channels by phosphorylation.
| |
FOOTNOTES |
Received July 17, 2002; revised Sept. 11, 2002; accepted Sept. 12, 2002.
This work was supported by grants from the National Institute of Mental
Health and the National Alliance for Research on Schizophrenia and
Depression (L.A.S., J.D.S.) and from the National Institute of
Neurological Disorders and Stroke (J.D.S., A.E.A.). We thank Dr. Dan
Johnston for helpful discussions.
Correspondence should be addressed to Dr. J. David Sweatt, Division of
Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. E-mail: jsweatt{at}bcm.tmc.edu.
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ABSTRACT
INTRODUCTION
