Neuron 50, 89–100, April 6, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.neuron.2006.03.010
The Kv4.2 Potassium Channel Subunit
Is Required for Pain Plasticity
Hui-Juan Hu,1Yarimar Carrasquillo,1Farzana Karim,1
Wonil E. Jung,2,5Jeanne M. Nerbonne,3
Thomas L. Schwarz,2and Robert W. Gereau IV1,4,*
1Washington University Pain Center and
Department of Anesthesiology
Washington University School of Medicine
St. Louis, Missouri 63110
Harvard Medical School
Boston, Massachusetts 02115
3Department of Molecular Biology and Pharmacology
Washington University School of Medicine
St. Louis, Missouri 63110
4Department of Anatomy and Neurobiology
Washington University School of Medicine
St. Louis, Missouri 63110
rons, A-type currents are modulated by extracellular
signal-regulated kinases (ERKs), which mediate cen-
tral sensitization during inflammatory pain. Here, we
report that Kv4.2 mediates the majority of A-type cur-
rent in dorsal horn neurons and is a critical site for
modulation of neuronal excitability and nociceptive
behaviors. Genetic elimination of Kv4.2 reduces
A-type currents and increases excitability of dorsal
horn neurons, resulting in enhanced sensitivity to tac-
tile and thermal stimuli. Furthermore, ERK-mediated
modulation of excitability in dorsal horn neurons and
ERK-dependent forms of pain hypersensitivity are ab-
sent in Kv4.22/2mice compared to wild-type litter-
mates. Finally, mutational analysis of Kv4.2 indicates
that S616 is the functionally relevant ERK phosphory-
lation site for modulation of Kv4.2-mediated currents
in neurons. These results show that Kv4.2 is a down-
stream target of ERK in spinal cord and plays a crucial
role in pain plasticity.
Chronic pain is an expression of neuronal plasticity,
which is mediated in part by increased excitability of no-
ciceptive neurons in thedorsal horn ofthe spinal cord(Ji
and Woolf, 2001). The molecular mechanisms that un-
but the extracellular signal-regulated kinases (ERKs)
have been implicated in the development of spinal cen-
tral sensitization underlying persistent pain (Adwanikar
et al., 2004; Galan et al., 2002; Hu and Gereau, 2003;
Hu et al., 2003; Ji et al., 1999, 2002, 2003; Ji and Woolf,
2001; Karim et al., 2001, 2006; Kawasaki et al., 2004;
Kominato et al., 2003; Lever et al., 2003; Pezet et al.,
2002). Although the precise cellular mechanisms of
ERK-dependent central sensitization are not known,
a number of mechanisms have been proposed, includ-
ing regulation of gene transcription and phosphoryla-
tion-dependent modulation of ion channels.
Among the potential ion channel targets, transient
outward (A-type) potassium channels have emerged
as attractive candidate sites of modulation for ERK-de-
pendent central sensitization of spinal cord dorsal horn
neurons (Hu and Gereau, 2003; Hu et al., 2003; Ji et al.,
2003; Karim et al., 2006). A-type K+ channels activate
at subthreshold membrane potentials, inactivate rap-
idly, and rapidly recover from inactivation. A-type cur-
rents are important regulators of neuronal excitability
and have been implicated in synaptic plasticity (Ram-
akers and Storm, 2002; Watanabe et al., 2002). In the
brain, A-type currents can be generated by Kv1.4,
Kv3.4, or any of the Kv4 family subunits (Kv4.1, Kv4.2,
and Kv4.3) (Pongs, 1999; Song, 2002). ERK activation
decreases A-type currents and increases excitability of
neurons in the superficial spinal cord dorsal horn (Hu
and Gereau, 2003; Hu et al., 2003; Karim et al., 2006)
and hippocampus (Watanabe et al., 2002; Yuan et al.,
2002). Furthermore, previous studies have demon-
strated that Kv4.2 is directly phosphorylated by ERKs
in vitro and in vivo (Adams et al., 2000). Since ERKs
play important roles both in nociception and modulation
of A-type currents, we hypothesized that the Kv4.2 sub-
unit might be a downstream target of ERK in dorsal horn
neurons that contributes to A-type currents and modu-
lates neuronal excitability and nociceptive behavior. Be-
cause of the dearth of pharmacologic reagents that tar-
get Kv4 channels, we have utilized dominant-negative
constructs and Kv4.2 knockout mice to directly test
Kv4 Channels Underlie A-Type K+ Currents
in Dorsal Horn Neurons
Kv4 channels are products of three distinct genes:
Kv4.1, Kv4.2, and Kv4.3 (Coetzee et al., 1999; Jerng
et al., 2004). To test whether these Kv4 subunits contrib-
ute to A-type currents in dorsal horn neurons, mouse
spinal cord superficial dorsal horn neurons were trans-
fected with an EGFP-tagged dominant-negative Kv4.2
construct (Kv4.2dn) containing a single amino acid sub-
stitution (W362F) (Barry et al., 1998) or with EGFP alone.
Using whole-cell voltage-clamp recordings in neurons
transfected with EGFP, large outward currents were
evoked by a step depolarization from a holding potential
reported previously in untransfected neurons (Hu et al.,
2003). Transfection with EGFP-Kv4.2dn dramatically re-
duced the A-type current (Figure 1A). Average A-type
current density in EGFP-Kv4.2dn-transfected neurons
was decreased by 79% compared to EGFP-transfected
controls, while sustained currents were not affected
(Figure 1B). Because the Kv4.2dn construct will disrupt
5Present address: Alertness Solutions, 20111 Stevens Creek Blvd,
Suite 280, Cupertino, California 95014.
channels containing any Kv4 subunit (Barry et al., 1998),
these results suggest that the Kv4 family is a prominent
contributor of A-type currents in dorsal horn neurons.
Consistent with this, immunostaining revealed strong
staining in the superficial layers of the spinal cord dorsal
horn using both anti-Kv4.2 and anti-Kv4.3 antibodies
(Figures 2B and 2C).
Kv4.2 Knockout Mice Reveal a Central Role of Kv4.2
in Regulating Neuronal Excitability of Spinal Dorsal
To test the hypothesis that Kv4.2 contributes to A-type
currents in superficial dorsal horn neurons, we exam-
ined A-type currents in dorsal horn neurons from mice
carrying a null mutation in the Kv4.2 gene that we have
generated by homologous recombination (Guo et al.,
2005). In the targeting construct, the principal exon,
which encodes most of the Kv4.2 channel (from the start
codon to Gly-373 in the middle of the pore domain), was
replaced with a neomycin cassette to create a null allele
that would neither give rise to a functional channel nor
produce a defective subunit that would dominantly in-
expression in the spinal cord of Kv4.22/2mice was ver-
ified by immunoblotting and immunocytochemistry us-
ing an anti-Kv4.2 antibody (Figures 2A and 2B). Kv4.3
protein was detected in both wild-type and Kv4.22/2
mice (Figure 2C).
To investigate whether deletion of Kv4.2 alters A-type
ficial dorsal horn neurons prepared from wt or Kv4.22/2
with the Kv4.2dn construct, the amplitude of A-type cur-
rents was decreased by 56% in Kv4.22/2neurons com-
pared to parallel cultures prepared from wt mice, while
sustained currents did not significantly differ between
genotypes (Figures 2D and 2E). In Kv4.22/2neurons,
the activation curve for A-type currents is significantly
shifted to the left by 6.5 mV with no change in slope
relative to wt (Figure 2F). The voltage of half-maximal
Figure 1. Kv4 Subunits Mediate A-Type K+
Currents in Spinal Cord Dorsal Horn Neurons
(A) Outward potassium currents recorded in
cultured mouse superficial dorsal horn neu-
rons transfected with control (EGFP, left
panel) or Kv4.2 dominant-negative (EGFP-
Kv4.2dn, right panel) constructs. The insets
show A-type currents after offline subtraction
of the sustained currents.
(B) Meandensities ofA-typecurrentsand sus-
tained currents in EGFP- or EGFP-Kv4.2dn-
mean 6 SEM; n = 12–16 neurons. **p < 0.01.
Figure 2. Genetic Elimination of Kv4.2 Dra-
matically Alters A-Type Currents in Dorsal
(A) Immunoblot of Kv4.2 protein from wt or
Kv4.22/2spinal cords. The position of molec-
ular weight markers is indicated.
(B and C) Immunohistochemical staining of
lumbar spinal cord sections from wt (left
panels) and Kv4.22/2mice (right panels) with
anti-Kv4.2 (B) or anti-Kv4.3 (C) antibodies.
(D) Representative A-type currents recorded
from wt or Kv4.22/2neuronal cultures.
tained currents in wt (n = 30) or Kv4.22/2(n =
44) neurons. Values represent mean 6 SEM;
***p < 0.001.
(F) Steady-state inactivation and activation
curves from wt (n = 7) or Kv4.22/2neurons
(n = 9).
(G) Recovery from inactivation of A-type cur-
rents in wt (n = 7) or Kv4.22/2(n = 8) neurons.
inactivation was not significantly different between the
two genotypes, but the slope of the steady-state inacti-
vation curve in neurons from Kv4.22/2neurons (19.5 6
2.0) was significantly different from that in wt neurons
(9.7 6 0.9, Figure 2F; p < 0.05, ANOVA). In addition,
recovery of A-type currents from inactivation was signif-
These changes in functional properties of A-type cur-
rents in Kv4.22/2mice were rescued by transfection of
dorsal horn neurons from Kv4.22/2mice with a wild-
type Kv4.2 construct (Figures 3A–3C), indicating that
the decrease in A-type currents observed in dorsal
horn neurons from Kv4.22/2mice is due to loss of Kv4.2
expression as opposed to an associated compensatory
change in expression of another gene. These results
suggest that Kv4.2 expression is necessary for the
majority of A-type currents in mouse superficial dorsal
Our previous studies showed that acute decreases in
A-type currents cause increases in neuronal excitability
in dorsal horn neurons (Hu and Gereau, 2003). As dele-
tion of Kv4.2 also reduces A-type currents relative to
wild-type in dorsal horn neurons, we hypothesized that
neuronal excitability would be enhanced in dorsal horn
neurons from Kv4.22/2mice relative to those from
wild-type mice. To test this hypothesis, we performed
whole-cell current-clamp recordings in superficial dor-
sal horn neurons from spinal cord slices prepared from
wt or Kv4.22/2mice. Lamina I/II neurons from Kv4.22/2
mice showed dramatically enhanced excitability com-
pared to neurons in slices from wt mice. In slices from
wt mice, the resting membrane potential was 266.4 6
1.6 mV (n = 28) and the mean rheobase (the minimum
current required to discharge an action potential) was
9.5 6 1.0 pA. Deletion of Kv4.2 significantly changed
the membrane potential to 262.2 6 1.2 mV (n = 25, p <
0.05) and significantly decreased the rheobase to
5.5 6 0.6 pA (Figure 4). During rheobase measurements,
neurons were always held at 270 mV via current injec-
tion.These results indicate
A-type currents regulate neuronal excitability in spinal
cord superficial dorsal horn neurons.
We and others have previously identified at least four
categories of superficial dorsal horn neurons based on
firing properties in culture and in slices (Hu and Gereau,
2003; Ruscheweyh and Sandkuhler, 2002). Similarly, in
the present study we identified neurons in spinal cord
slices from ICR mice or wild-type 129SvEv mice that in-
clude a mixture of repetitive (tonic), phasic, and delayed
firing patterns. Interestingly, Kv4.22/2mice show a
change in the distribution, with a near absence of phasic
firing cells andacoincident increase inthe groupofcells
showing repetitive firing in response to a step current in-
jection (Table 1). This is similar to previous results ob-
tained in superior cervical ganglion neurons transfected
with a Kv4.2 dominant-negative construct (Malin and
Nerbonne, 2001) and suggest that Kv4.2-containing
channels may contribute to the currents that mediate
spike frequency adaptation in dorsal horn neurons. Al-
ternatively, the absence of Kv4.2 may lead to compen-
satory changes in other ion channels that normally gen-
erate this phasic firing pattern in dorsal horn neurons.
The increase in the percentage of tonic-firing neurons
suggests that the neurons that have a phasic firing
pattern in wild-type animals have been converted to
a tonic-firing pattern in Kv4.22/2mice. The precise
mechanisms of this change are not known.
Altered Nociception in Kv4.2 Knockout Mice
Theresults describedabove demonstrate thatKv4.2un-
derlies the majority of A-type current in spinal dorsal
horn neurons, and accordingly dorsal horn neurons
from Kv4.22/2mice show increased excitability relative
to neurons from wt mice (Figure 4). Because superficial
dorsal horn neurons are involved in nociceptive trans-
mission, we hypothesized that the altered neuronal ex-
citability in Kv4.22/2mice would result in altered pain
transmission in these animals. To test this hypothesis,
we evaluated nociceptive behavior in Kv4.22/2mice
and their wt littermates (Kv4.2+/+). All behavioral studies
Figure 3. Rescue of A-Type Currents in Kv4.22/2Neurons by Trans-
fection with a Wild-Type Kv4.2 Construct
fected with EGFP or EGFP + Kv4.2.
(B) Mean densities of A-type currents and sustained currents in
Kv4.22/2neurons transfected with EGFP (n = 20) or EGFP + Kv4.2
(n = 22). Values represent mean 6 SEM; ***p < 0.001.
(C) Recovery from inactivation of A-type currents recorded in
Kv4.22/2neurons transfected with EGFP (n = 6) or EGFP + Kv4.2
(n = 8).
Kv4.2 in Spinal Cord Pain Processing
wereperformed prior to genotyping, and thus theexper-
imenter was always blind to genotype. Kv4.22/2mice
were indistinguishable from sex- and age-matched
wild-type littermates in appearance, general behavior,
andbody weight(datanot shown) (Guoetal.,2005). Fur-
thermore, motor function assessed using an accelerat-
ing rotarod was normal (Figure 5A). However, consistent
with our hypothesis, Kv4.22/2mice displayed signifi-
cantly enhanced sensitivity to mechanical and thermal
stimuli compared to Kv4.2+/+littermates. The paw-
withdrawal threshold to von Frey filament stimulation
and the response threshold to noxious pressure applied
to the tail were significantly reduced in Kv4.22/2mice
compared to their Kv4.2+/+littermates (Figures 5B and
5C). Kv4.22/2mice also had significantly shorter with-
drawal latencies to noxious heat in the hotplate test
when compared to their Kv4.2+/+littermates (Figure 5D).
ERK-Dependent Modulation of Dorsal Horn Neuronal
Excitability Requires Kv4.2
Our previous studies showed that activation of ERK
causes significant inhibition of A-type currents and ac-
cordingly increases neuronal excitability in mouse dor-
sal horn neurons (Hu and Gereau, 2003; Hu et al.,
2003). Given that the intracellular C terminus of Kv4.2
can serve as an ERK substrate in vitro (Adams et al.,
of A-type currents and neuronal excitability in dorsal
horn neurons might require Kv4.2 expression. To test
this hypothesis, we compared the effects of ERK activa-
tion and inhibition in dorsal horn neurons from wt and
There are no specific pharmacologic agents that acti-
vate the ERK pathway independent of other kinases.
However, activation of PKA or PKC can lead to down-
stream activation of MEK/ERK signaling. We previously
showed that all of the effects of the PKC activator PMA
on dorsal horn neuronal physiology are blocked by the
MEK inhibitors U0126 and PD98059, suggesting that
these effects of PMA are due to activation of ERK down-
therefore tested the effects of PMA (5 mM) as an up-
stream activator of ERK, or the MEK inhibitor PD98059
(20 mM), which inhibits activation of ERK, on cultured
dorsal horn neurons or lamina I/II neurons in spinal
cord slices. Consistent with our previous report (Hu
et al., 2003), in cultured dorsal horn neurons from wt
mice, PMA significantly reduced A-type current ampli-
tude, while PD98059 increased the current amplitude.
In the Kv4.22/2neurons, however, PMA had no effect
on A-type currents, and PD98059 did not increase but
rather decreased A-type currents (Figures 6A and 6B).
These results demonstrate that the inhibition of A-type
currents by PMA and the PD98059-induced increases
in these currents require expression of the Kv4.2 sub-
unit. The inhibitory effect of PD98059 on A-type currents
in Kv4.22/2neurons compared to the enhancement of
A-type currents by PD98059 in wt neurons could be
due to an inhibitory modulation by ERK signaling of
the residual current in the Kv4.22/2neurons or could
represent a nonspecific effect of the drug.
In current-clamp recordings in slices prepared from
wt mice, PMA significantly decreased first-spike latency
and increased spike frequency, while PD98059 signifi-
cantly increased the first-spike latency and reduced
the spike frequency. These effects of ERK activation
by PMA, or inhibition by PD98059, were completely ab-
sent in slices prepared from Kv4.22/2mice (Figures 6C
and 6D). These results indicate that expression of the
Kv4.2 subunit is necessary for ERK-dependent modula-
tion of neuronal excitability in spinal cord dorsal horn
Loss of ERK-Dependent Hyperalgesia
in Kv4.2 Knockout Mice
The behavioral data to this point showing hypersensitiv-
ity to tactile and thermal stimuli are completely consis-
tent with our observations of enhanced excitability of
dorsal horn neurons in the Kv4.22/2mice. A further pre-
diction, based on our observation that ERK-dependent
modulation of A-type currents and neuronal excitability
in dorsal horn neurons requires Kv4.2 expression, is
Figure 4. Genetic Elimination of Kv4.2 Re-
sults in Increased Excitability of Dorsal Horn
(A) Representative action potentials gener-
ated by an increasing series of current injec-
tions (left panel) recorded in superficial (lam-
ina I-II) dorsal horn neurons in spinal cord
slices prepared from wt (center panel) or
Kv4.22/2(right panel) mice.
(B) Rheobase (the minimum current required
to elicit an action potential) in neurons from
wt (n = 28) orKv4.22/2(n = 25) mice.For rheo-
base measurements, neurons were always
held at 270 mV via current injection. Values
represent mean 6 SEM; **p < 0.01.
Table 1. Firing Properties of Dorsal Horn Neurons from Spinal Cord Slices
MouseRepetitive PhasicDelayed Firing Single Spike
that ERK-dependent forms of behavioral sensitization
involving central sensitization of dorsal horn neurons
should also be absent in these animals. The prediction
is that the Kv4.22/2mice, while hypersensitive in the
basal condition due to increased excitability of dorsal
horn neurons, would not develop further hypersensitiv-
ity that results from ERK-dependent modulation in dor-
sal horn neurons. To test this hypothesis, we compared
Kv4.22/2mice to wild-type littermates in the formalin
and carrageenan models of inflammatory pain and in
the chronic constriction injury (CCI) model of neuro-
pathic pain, all of which have been shown to involve
ERK activation in the spinal dorsal horn (Ciruela et al.,
2003; Galan et al., 2002; Ji et al., 1999; Karim et al.,
2001; Song et al., 2005).
The formalin test is commonly employed as a test of
inflammatory pain in rodents. Injection of formalin into
the hindpaw activates nociceptors and results in a typi-
cal biphasic nociceptive response (Karim et al., 2001).
The first phase of nocifensive behaviors is generally
believed to involve direct activation of nociceptors,
whereas the second phase is believed to additionally in-
volve peripheral and central sensitization (Puig and Sor-
kin, 1996). This sensitization in the second phase has
been shown by multiple groups to require ERK activa-
tion in the spinal dorsal horn (Ji et al., 1999; Karim
et al., 2001). We therefore compared the response of
Kv4.22/2mice and Kv4.2+/+littermates in the formalin
test. For this experiment, we utilized mice backcrossed
onto the FVB background for >12 generations, as the
129SvEv strain of mice expressed very little formalin-in-
duced behaviors in the second phase (data not shown),
consistent with previous reports (Mogil et al., 1999).
No significant difference between Kv4.22/2mice and
Kv4.2+/+littermates was detected in the acute phase
(Figure 7A), but the second phase behavioral response
in the Kv4.22/2mice is very interesting. These mice
show a constant mild elevated response compared to
the Kv4.2+/+mice; however, they lack the peak second
phase response observed in the Kv4.2+/+littermates
(Figure 7A). While an ANOVA comparing the traditionally
defined formalin second phase (15 min to 60 min post-
formalin) did not reach significance, a significant differ-
ence is observed between Kv4.22/2mice and Kv4.2+/+
littermates when considering nociceptive behaviors
10–30 min after formalin injection (repeated-measures
ANOVA). There is no significant difference between ge-
notypes for the 35–45 min block. The constant mild ele-
vated second phase response observed in Kv4.22/2
mice is consistent with the idea that there is enhanced
postsynaptic firing (due to enhanced excitability of dor-
sal horn neurons in Kv4.22/2animals) in response to the
malin second phase (Puig and Sorkin, 1996), whereas
the lack of a peak second phase in the Kv4.22/2mice
can be attributed to the lack of ERK-dependent modula-
tion of excitability in these superficial dorsal horn neu-
rons. Interestingly, these results in the formalin test are
reminiscent of our results of studies of transgenic mice
that express dominant-negative MEK in neurons, in
which we observed a reduction in only the first half of
We next tested for the presence of mechanical hyper-
sensitivity 1–3 hr following formalin injections. Kv4.2+/+
mice developed significant ipsilateral and contralateral
hypersensitivity to mechanical stimuli, while Kv4.22/2
littermates were not hypersensitive relative to baseline
mechanical thresholds (Figure 7B), suggesting that
Kv4.2 is required for inflammation-induced mechanical
hypersensitivity. While previous studies have reported
enhanced nociceptive sensitivity following formalin-in-
duced inflammation (Fu et al., 2000, 2001; Zeitz et al.,
2004), no studies have addressed whether this hyper-
sensitivity is dependent on spinal ERK signaling. We
therefore investigated whether the absence of mechan-
ical hypersensitivity in Kv4.22/2mice was due to a lack
of ERK-dependent modulation in the spinal cord. We
pretreated wt mice with the MEK inhibitor U0126 (2
nmol, intrathecal) for 15 min, then injected formalin
into the hindpaw. U0126 application did not alter basal
mechanical thresholds, but significantly reduced forma-
lin-induced mechanical hypersensitivity (Figures 7C and
7D). These results suggest that tactile hypersensitivity
after formalin-induced inflammation requires ERK acti-
vation in the spinal cord, and further implicates ERK-
mediated modulation of Kv4.2-containing channels in
this process. It is important to point out that we used
mice from several different genetic backgrounds
(129SvEv, FVB, and ICR) in this study. Although the ab-
solute mechanical withdrawal thresholds were different
between the strains, the Kv4.22/2mice were consis-
tently more sensitive to touch at baseline than wild-
type littermates for a given strain (compare 129 mice in
Figure 5. Enhanced Basal Pain Behavioral Responses in Kv4.22/2
All values represent mean 6 SEM.
(A) Drop latencies on an accelerating rotarod (n = 14–16).
(B) Paw-withdrawal thresholds to von Frey filaments (n = 8–11).
(C) Response threshold to tail pressure (n = 14–16).
(D) Response latencies in the hot-plate test at 52ºC or 56ºC (n = 14–
*p < 0.05, versus wild-type, ANOVA. All mice used for behavioral
studies in this figure were on the 129 genetic background.
Kv4.2 in Spinal Cord Pain Processing
Figure 5 and Figure 7B to the FVB mice in Figure 7E and
Figure S1). Mice used in Figures 7C and 7D were ICR
The carrageenan model of inflammatory pain in rats
and mice is also associated with increased ERK activa-
tion in the spinal dorsal horn (Galan et al., 2002). In the
carrageenan model, we found a phenotype in the
Kv4.22/2mice that is very similar to what we observed
in the formalin model. Thus, while Kv4.22/2mice were
hypersensitive to touch relative to Kv4.2+/+mice prior
to carrageenan injection in the hindpaw, following in-
flammation the Kv4.2+/+mice developed hypersensitiv-
ity to touch in both the ipsilateral and contralateral
paw, whereas the mechanical-withdrawal thresholds in
Kv4.22/2mice did not differ from baseline for the ipsilat-
eral or contralateral paw (Figure 7E).
In addition to the above-mentioned models of inflam-
matory pain, the CCI model of neuropathic pain has
been shown to be associated with increased spinal
ERK activity, and intrathecally administered MEK inhib-
itors reduce allodynia in this model at concentrations
that are effective at inhibiting ERK activity (Ciruela
et al., 2003). Kv4.22/2mice and Kv4.2+/+littermates
both developed mechanical hypersensitivity following
CCI surgery (see Figure S1 in the Supplemental Data).
Interestingly, Kv4.22/2and Kv4.2+/+mice reached iden-
tical absolute withdrawal thresholds following nerve in-
jury. However, withdrawal thresholds in Kv4.2+/+mice
remained significantly reduced relative to pre-CCI base-
lines for >35 days after surgery, whereas in Kv4.22/2
mice, withdrawal thresholds returned to prebaseline
sents a physiologically significant effect is not clear. In
total, our behavioral data point to a more critical role
for ERK modulation of Kv4.2 in inflammatory pain.
Phosphorylation Site S616 Mediates
ERK Modulation of Kv4.2
Our working model is that Kv4.2 is directly phosphory-
lated by ERK, and this mediates a component of central
sensitization underlying inflammatory pain. As men-
teins identified three sites that were phosphorylated on
the Kv4.2 intracellular domains by ERK in vitro (Adams
etal.,2000).However, todatethereareno datasuggest-
ing which, if any, of these sites are important for ERK-
dependent modulation of Kv4.2-mediated currents in
neurons, and it is formally possible that this modulation
is dependent on ERK phosphorylation of an accessory
protein rather than Kv4.2 itself. To begin to address
Figure 6. Kv4.2 Is Required for ERK-Depen-
dent Modulation of A-Type K+ Currents and
Neuronal Excitability in Spinal Cord Dorsal
(A) Representative A-type currents recorded
before (pre-drug, black traces) and after ap-
plication of PMA (5 mM, blue traces) or
PD98059 (20 mM, red traces) to wt or
(B) Grouped data showing the effect of PMA
and PD98059 on peak amplitude of A-type
currents in wt (open bars) and Kv4.22/2
(hatched bars) neurons. Values represent
mean 6 SEM, n = 6–9 neurons. ***p < 0.001,
t test comparing genotypes.
corded in wt and Kv4.22/2dorsal horn neu-
rons (lamina I-II) of spinal cord slices before
(pre-drug) and after application of PMA
(5 mM) or PD98059 (20 mM).
(D) Effect of PMA (blue) and PD98059 (red) on
first-spike latency and spike frequency in wt
and Kv4.22/2neurons. Circles show individ-
ual values measured before (open circles) or
after (closed circles) drug application. Bars
represent mean 6 SEM of all cells; n = 5–10.
*p < 0.05, paired Students’ t test.
x For experiments in panels (C) and (D), cur-
rent injection amplitudes were adjusted to
elicit roughly equivalent firing patterns in the
wt and Kv4.22/2neurons in the pre-drug con-
ditions to allow direct comparison of the drug
effects in the different genotypes. Thus, the
baseline firing properties are not representa-
tive of differences between the genotypes
when equivalent currents are injected, as
shown in Figure 4.
this issue, we generated point mutations in each of the
three biochemically identified ERK phosphorylation
sites on Kv4.2 and used these to determine which of
the sites (if any) mediates ERK-dependent modulation
of Kv4.2. We transfected dorsal horn neurons from
Kv4.2 knockout mice with either wild-type Kv4.2 or ala-
nine mutants of the ERK phosphorylation sites T602,
T607, or S616 (Adams et al., 2000). Transfection of
Kv4.22/2dorsal horn neurons with wild-type Kv4.2 re-
stored A-type currents, and these currents were in-
hibited via an ERK-dependent mechanism by PMA, sim-
ilar to what we observed in wild-type neurons (Figures
8A and 8B and see Figures 6A and 6B). A-type currents
were also restored in Kv4.22/2dorsal horn neurons
transfected with the T602A, T607A, and S616A ERK
phosphorylation site mutants, and biophysical proper-
ties of these currents were indistinguishable from those
rons transfected with Kv4.2(T602A) or Kv4.2(T607A),
ERK-dependent inhibition of A-type currents was similar
Figure 7. Reduced ERK-Dependent Pain
Plasticity in Kv4.22/2Mice
(A) Formalin induced spontaneous nocicep-
tive behavior in FVB mice (n = 16–18).
(B) Time course of formalin-induced mechan-
ical allodynia (n = 8–18) inthe injected (ipsilat-
eral) paw or the noninjected (contralateral)
paw relative to baselines taken before injec-
tion of formalin (dashed line) in 129 mice.
(C) Effect in wt ICR mice of i.t. injection of
U0126 (2 nmol) on basal mechanical thresh-
olds (n = 10 each).
(D) Effect in wt ICR mice of i.t. injection of
U0126 (2 nmol) on formalin-induced mechan-
ical allodynia measured 1 hr after formalin in-
jection (n = 9–10). *p < 0.05.
(E) Carrageenan-induced mechanical hyper-
sensitivity in Kv4.22/2mice and wild-type lit-
termates (FVB mice, n = 5–6).
For all figures, all values represent mean 6
symbols and bars represent Kv4.22/2results,
open symbols and bars represent wild-type
Kv4.2+/+and Kv4.22/2from 10–30 min, 35–
45 min, and 50–60 min time periods are not
significantly different. ANOVA for total forma-
lin second phase (10–60 min) was not signifi-
yp < 0.05, ANOVA, comparing
Kv4.2 in Spinal Cord Pain Processing
to that observed in neurons transfected with wild-type
Kv4.2. In contrast, ERK-dependent modulation of
fected with Kv4.2(S616A) (Figure 8).
In the present study, we identify the Kv4.2 subunit as
a prominent contributor to A-type currents in dorsal
horn neurons and demonstrate that this subunit is a crit-
icalsiteofregulation inpainprocessing. Kv4.2knockout
mice have reduced A-type currents in dorsal horn neu-
rons, and this leads to increased excitability of these
neurons associated with a slight membrane depolariza-
tion. These lamina I-II neurons receive synaptic input
from primary afferent nociceptors, and one would hy-
pothesize that because of these alterations to neuronal
excitability, a small synaptic input would elicit more ac-
tion potentials in dorsal horn neurons from Kv4.22/2
mice than in those from wild-type mice. Accordingly,
we observed an increased sensitivity to touch and
heat in Kv4.22/2mice relative to their Kv4.2+/+litter-
mates. We also show that ERK-dependent modulation
of neuronal excitability found in wild-type dorsal horn
neurons is completely absent in dorsal horn neurons
from Kv4.22/2mice. Consistent with this absence of
ERK-dependent modulation of neuronal excitability,
we find that Kv4.22/2mice have significant deficits in
several pain behavioral models in which ERK signaling
has been implicated. Taken together, our results sug-
gest that Kv4.2-containing K+ channels represent a crit-
ical node of modulation that regulates transmission of
nociceptive signals from the periphery to the brain.
Because Kv4.2 is a known substrate for ERK phos-
phorylation (Adams et al., 2000), it is reasonable to hy-
pothesize that the reduction in ERK-dependent behav-
ioral sensitization in Kv4.2 knockout mice is due to the
requirement for ERK-mediated phosphorylation and in-
hibition of Kv4.2-containing K+ channels, which nor-
mally leads to enhanced excitability of superficial dorsal
horn neurons (Hu and Gereau, 2003; Hu et al., 2003;
Karim et al., 2006). The results of our analysis of ERK
phosphorylation site mutants of Kv4.2 are consistent
with this hypothesis and suggest that S616 is the func-
tionally relevant phosphorylation site on Kv4.2. A direct
test of this model would be a genetically modified
mouse that carries a mutation in the S616 ERK phos-
phorylation site of Kv4.2. The prediction would be that
these animals would have relatively normal A-type cur-
rents in dorsal horn neurons but no modulation of this
current by ERK activity. At the behavioral level, the pre-
diction would be that these mice would have normal
baseline pain sensitivity but have reduced hypersensi-
tivityininflammatory painmodels. Thiswould beslightly
different than what we observe in the Kv4.22/2animals,
which have reduced A-type currents and resultant hy-
perexcitability of dorsal horn neurons, leading to noci-
ceptive hypersensitivity under baseline conditions. In
contrast, the ERK-dependent behavioral hypersensitiv-
ity that occurs following inflammation should be absent
both in the Kv4.22/2mice and in the Kv4.2 ERK phos-
phorylation site mutant mice.
Although the model described above can account for
the behavioral differences observed in Kv4.22/2mice
relative to wild-type mice, it is formally possible that be-
cause the Kv4.22/2animals are hypersensitive under
baseline conditions, they have simply reached their
maximum level of sensitivity. For several reasons, we
believe that this is not the case. First, the withdrawal
thresholds for wild-type mice after formalin- or carra-
geenan-induced inflammation are significantly lower
than the Kv4.22/2mice in the basal state and after
Figure 8. The S616 Phosphorylation Site Me-
diates ERK Modulation of Kv4.2-Mediated
A-Type Currents in Spinal Cord Dorsal Horn
(A) Representative A-type currents recorded
neurons transfected with
EGFP + wild-type Kv4.2 or EGFP + Kv4.2
phosphorylation site mutants (T602A, T606A,
or S616A) before (pre-drug, black traces)
and after application of PMA (5 mM, red
traces). The MEK inhibitor PD98059 (20 mM)
blocks PMA-induced modulation of A-type
currents transfected with wild-type Kv4.2,
as well as those transfected with T602A or
T607A (not shown).
EGFP + wild-type Kv4.2 or + Kv4.2 phosphor-
ylation site mutants. Values represent mean 6
with control (vehicle).
(C) Steady-state inactivation and activation
curves from Kv4.22/2neurons transfected
with EGFP + wild-type Kv4.2 or + Kv4.2 phos-
phorylation site mutants (n = 8–14).
(D) Recovery from inactivation of A-type cur-
rents in Kv4.22/2neurons transfected with
EGFP + wild-type Kv4.2 or + Kv4.2 phosphor-
ylation site mutants (n = 8–9).
inflammation (Figures 7B and 7E). Second, the Kv4.22/2
mice are clearly able to be made more hypersensitive to
touch, as there is significant hypersensitivity in the
lines (Figure S1). These data are consistent with the hy-
pothesis that the lack of hypersensitivity in the Kv4.22/2
mice relative to Kv4.2+/+littermates is due to reduced
plasticity in the Kv4.22/2mice and not due to a behav-
ioral ‘‘floor effect.’’
The finding that Kv4.22/2mice still have some hyper-
sensitivity in the CCI model of persistent neuropathic
pain is interesting given the diversity of actions of ERK
signaling in neurons (Ji et al., 2003). It is possible that
ERK phosphorylation and modulation of Kv4.2-contain-
ing K+ channels mediates a component of central sensi-
tization but that transcription-dependent changes un-
derlie other long-term components of ERK-dependent
central sensitization that are not reduced in the Kv4.2
knockouts (Ji and Rupp, 1997; Ji et al., 2002; Woolf
and Costigan, 1999). Our data are consistent with the
hypothesis that Kv4.2 modulation plays a more sig-
nificant role in inflammatory pain than in longer-term
neuropathic pain conditions, where ERK-dependent
transcriptional regulation may be more important. The
Kv4.22/2animals and potential phosphorylation site
mutant animals may provide useful tools for future stud-
ies examining the relative importance of acute phos-
phorylation and modulation of K+ channels and tran-
scription/translation-dependent changes in mediating
various forms of long-term pain hypersensitivity.
Although our data are consistent with a critical role of
ERK modulation of Kv4.2-containing potassium chan-
nels in superficial spinal dorsal horn neurons in mediat-
ing behavioral sensitization following inflammation, it is
certainly true that Kv4.2 is expressed in many areas of
the nervous system. Thus, it is possible that Kv4.2 plays
important roles in other parts of the pain neuraxis and
a component of the alterations in behavior observed in
iology in these areas in addition to the spinal cord. We
were particularly curious to see whether Kv4.2 might
be impacting the physiology of nociceptive primary
afferent neurons, but we were unable to detect any
Kv4.2 expression in the mouse dorsal root ganglion by
ficial laminae of the spinal cord contain both excitatory
and inhibitory neurons, and excitatory neurons can be
projection neurons or interneurons. Because we do
not know the transmitter phenotype of all of the neurons
from which we have recorded, the specific cellular cir-
cuitry impacted by these changes remains to be deter-
Our results reveal a link between Kv4.2 and ERK sig-
naling in the spinal cord and support a model in which
Kv4.2-containing potassium channels in spinal super-
ficial dorsal horn neurons are modulated by ERKs to in-
duce a component of central sensitization. Our data fur-
ther implicate direct phosphorylation of Kv4.2 at S616 in
this process. ERK-dependent phosphorylation of Kv4.2
and modulation of A-type K+ currents appears to under-
lie plasticity in hippocampal pyramidal neurons asso-
ciated with long-term potentiation (LTP) (Kim et al.,
has been considerable interest in the parallels between
LTP and central sensitization (Basbaum, 1996; Ji et al.,
2002); the present study supports the idea that LTP and
central sensitization may share common mechanisms,
as our studies and previous work have demonstrated
the importance of ERK signaling and Kv4.2 in both of
these processes (Ji et al., 2003). Whether these similar-
sensitization is worthy of extensive study.
The results presented here advance our understand-
ing of the mechanisms underlying pain processing and
are especially relevant to our understanding of inflam-
mation-induced pain hypersensitivity. Our findings sug-
gest that manipulation of the ERK-Kv4.2 signaling path-
way could be useful for novel therapies for the treatment
Cell Culture and Spinal Cord Preparation
Primary cultures of spinal cord superficial dorsal horn neurons were
prepared from 4- to 8-day-old CD1 mice and 129SvEv wild-type or
Kv4.22/2mice as previously described (Hugel and Schlichter,
2000). Lumbar spinal cord slices (300–350 mm) were prepared from
6- to 10-day-old CD1 or 129SvEv mice as previously described (Ed-
wards et al., 1989) and maintained in artificial cerebrospinal fluid
(ACSF) containing (in mM) 118 NaCl, 3 KCl, 24 NaHCO3, 2 MgCl2,
1.25 NaH2PO4, 1 CaCl2, and 12 glucose at room temperature under
continuous oxygenation for 1–4 hr.
Transfection of Dorsal Horn Neurons
The EGFP-Kv4.2dn construct was generously provided by Dr. Paul
Pfaffinger (Baylor College of Medicine). Plasmid DNA was isolated
using the Qiagen plasmid maxi protocol. Spinal cord dorsal horn
neurons were cultured for 24 hr and then transfected with plasmid
DNA constructs (0.45–0.9 mg/well) overnight using LipofectAMINE
Plus or 2000 reagent according to the manufacturer’s protocol (In-
vitrogen Carlsbad, CA). Patch-clamp recordings were performed
beginning w16 hr following transfection. Point mutations in ERK
phosphorylation sites were generated as described previously (Ger-
eau and Heinemann, 1998).
Whole-cell recordings were performed using standard procedures
at room temperature using either an AXOPATCH 200B amplifier
and CLAMPEX 8.0 software (Axon Instruments, Union City, CA) or
an EPC-10 amplifier and Pulse v8.62 software (HEKA Elektronik,
Lambrecht, Germany) as previously described (Hu and Gereau,
2003; Hu et al., 2003). Electrode resistances were 3–6 MU with series
resistances around 6–15 MU and were compensated by R60%. For
voltage-clamprecordings inculturedneurons, the bath solution was
Hank’s solution (HBSS) (in mM: 137 NaCl, 5.4 KCl, 0.4 KH2PO4,
1 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 4.2 NaHCO3, 0.3 Na2HPO4, 5.6 glu-
cose) containing 500 nM TTX and 2 mM CoCl2to block voltage-
gated Na+currents, Ca2+currents, and Ca2+-activated K+ currents.
The electrode solution contained (in mM) 140 KCl, 1 MgCl2,
0.5 CaCl2, 5 EGTA, 10 HEPES, 3 Na2ATP, and 0.3 Na2GTP (pH 7.4).
The membrane voltage was held at 280 mV, and transient po-
tassium currents (IA) were isolated by a two-step voltage protocol
as previously described (Hu et al., 2003). To determine the voltage-
dependent activation,voltage stepsof 500mswereapplied at 5sin-
tervals in +10 mV increments from 270 mV to a maximum of +70 mV.
To determine the voltage-dependent inactivation, conditioning pre-
pulses ranging from –120 mV to +40 mV were applied at 5 s intervals
in +10 mV increments for 150 ms followed by a step to +40 mV for
500 ms. To determine time-dependent recovery from inactivation,
conditioning pulses (40 mV) were applied for 500 ms followed by
steps to +40 mV for 500 ms in 20 or 200 ms incremental duration.
For current-clamp recording in slice, the bath solution was ACSF
bubbled with 95% O2-5% CO2. Intracellular solution contained
Kv4.2 in Spinal Cord Pain Processing
Na2GTP (pH 7.4). Action potentials were generated by current injec-
tion from a holding potential of 270 mV for rheobase or 277 mV for
Generation of Kv4.2 Knockout Mice
Kv4.2-deficient mice were generated as we have previously de-
scribed (Guo et al., 2005). Briefly, the targeting vector was con-
structed in the pPNT vector using 129Sv genomic DNA from a phage
isolate. For the 30arm, a 7.1 kb AflII genomic fragment, downstream
of the first coding exon, was inserted into a SrfI site that previously
been introduced between the KpnI and EcoRIsites of the vector. For
the 50arm, a 3.1 kb SpeI-ScaI genomic fragment upstream of the
predicted start codon was inserted between the NotI and the XhoI
sites. Thus, the coding region of the first exon of Kv4.2 was replaced
with the neor gene. ES cells (gift of Dr. Andras Nagy, University of
Toronto) were electroporated with the targeting vector and selected
with G418 and gancyclovir. Clones were injected into blastocysts to
generate chimeric mice, which were crossed to 129/SvEv mice to
produce Kv4.2+/2mice.Wild-type and Kv4.22/2littermate mice gen-
Western Blot Analysis
The lumbar section of the spinal cord was dissected from adult
129SvEv wild-type or Kv4.22/2mice and homogenized using
homogenization buffer (HB) (Tris 20 mM, pH 7.5, EDTA 1 mM,
Na4P2O71 mM, aprotinin 25 mg/ml, leupeptin 25 mg/ml, Na3VO4
0.2 mg/ml, and PMSF 0.4 mM). Membrane proteins were separated
and electrophoresed in 10% SDS polyacrylamide gels. The blots
were blocked with 5% milk and probed with anti-Kv4.2 primary
antibody (1:500, Chemicon). The blots were incubated with HRP-
conjugated secondary antibody (1:20,000, Cell Signaling) and devel-
oped with the SuperSignal West Femto reagent (Pierce).
Thirty-micron sections of paraformaldehyde-fixed lumbar spinal
cord from adult 129SvEv wild-type or Kv4.22/2mice were stained
with rabbit anti-Kv4.2 (1:800, Alomone) or rabbit anti-Kv4.3 primary
antibody (1:200, Chemicon). Sections were then incubated in bioti-
nylated secondary antibody (1:200, Vector) and treated in extrAvidin
peroxidase (1:1000,Sigma). Detectionwasperformedusingadiami-
nobenzidine (DAB) substrate kit (Vector).
All behavioral tests were performed blind using 7- to 9-week-old
mice. All the experiments were done in accordance with the guide-
lines of the National Institutes of Health and The International Asso-
ciation for the study of Pain and were approved by the Animal Care
and Use Committee of Washington University School of Medicine.
Wild-type and Kv4.22/2littermate mice of both sexes were tested
except where mentioned.
Mice were tested for motor function using the accelerating rotarod
(4–40 rpm) (UGO Basile, Varese, Italy). The time spent on the rotarod
Basal Sensitivity to Mechanical and Thermal Stimulation
aments (North Coast Medical, Inc., San Jose, CA) as previously de-
scribed (Yang and Gereau, 2003). The smallest monofilament that
evoked paw-withdrawal responses on three out of five trials was
taken as the mechanical threshold. The tail pressure test was con-
ducted using the Basile Analgesy-Meter, and the response (strug-
gle/vocalization) threshold was measured as previously described
(Nassar et al., 2004). Hot plate latencies were measured as the
time taken for a mouse to lick or shake its hindpaw at hot plate tem-
peratures of either 52ºC or 56ºC.
The formalin test was performed by injection of 5% formalin subcu-
spent in spontaneous nociceptive behavior (licking and lifting of the
injected paw) was recorded in 5 min intervals for 1 hr as previously
described (Karim et al., 2001). Mechanical allodynia was measured
in male mice from 1–3 hr after injection of formalin using von Frey
filaments. For experiments analyzing the effect of U0126 on forma-
ter formalin injection. Biochemical studies were performed to test
the optimal dose and timing for U0126 intrathecal injections and
revealed that intrathecal injection of 2 nmol of U0126 was able to in-
hibit ERK activation for up to 2 hr after intrathecal injection (data not
shown). Based on this finding, U0126 was injected 15 min prior to
formalin injection, and allodynia was measured 1 hr after formalin
Two percent carrageenan was injected subcutaneously into the
plantar surface of the right hindpaw. Mechanical hypersensitivity
was measured inmale mice 2hr after injection of carrageenan, using
von Frey filaments as described above.
Chronic Constrictive Injury Model
CCI of the sciatic nerve was induced as previously described (Ben-
dium anesthesia, the left sciatic nerve was exposed at mid-thigh,
and two ligatures were loosely ligated proximal to the sciatic’s
trifurcation at w1.0 mm intervals with 6-0 chromic gut sutures. Me-
chanical hypersensitivitywas measuredon 1,4,7,10, 14, 21, 28, and
35 days post-CCI, using von Frey filaments as described above.
Stock solutions of phorbol 12-myristate 13-acetate (PMA), PD
098059 (Sigma-Aldrich, St. Louis, MO), and U-0126 (Calbiochem,
La Jolla, CA) were made in DMSO and diluted to final concentrations
in HBSS for bath applications or in PBS for intrathecal injections.
Offline evaluation was done using clampfit 8.0 (Axon Instrument) or
Pulse v8.62 (HEKA) and Origin (Microcal Software Inc., Northamp-
ton, MA). Data are expressed as original traces and/or as mean 6
SEM. The voltage dependence of activation and inactivation of the
IAwas fitted with the Boltzmann function as previously described
(Hu et al., 2003). Behavioral experiments were statistically analyzed
by ANOVA (or repeated-measures ANOVA when appropriate as indi-
cated) followed by the appropriate post hoc tests. Paired or two-
to two means. Error probabilities of p < 0.05 were considered statis-
The Supplemental Data for this article can be found online at http://
The authors wish to thank P. Pfaffinger for generously providing the
Kv4.2dn construct; and C. Qiu, S. Shalin, and B. McGill for help with
mouse colony maintenance, immunostaining, and dorsal horn neu-
from the NIH (R.W.G., Y.C., J.M.N., and T.L.S.), Arthritis Foundation
(F.K.), and the Paralyzed Veterans of America Spinal Cord Research
Received: July 8, 2005
Revised: August 10, 2005
Accepted: March 3, 2006
Published: April 5, 2006
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