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Am J Physiol Cell Physiol 305: C197–C206, 2013.
First published May 22, 2013; doi:10.1152/ajpcell.00041.2013.
Neuregulin-1/ErbB4 signaling regulates Kv4.2-mediated transient outward
K⫹ current through the Akt/mTOR pathway
Jin-Jing Yao,* Ji Sun,* Qian-Ru Zhao, Chang-Ying Wang, and Yan-Ai Mei
State Key Laboratory of Medical Neurobiology, School of Life Sciences and Institutes of Brain Science, Fudan University,
Shanghai, China
Submitted 6 February 2013; accepted in final form 13 May 2013
neuregulin; ErbB4; Akt/mTOR; Ia; Kv4.2; CGNs
member of a family of neurotrophic
factors that contain EGF-like repeats and bind to and activate
EGF family receptors (ErbB2– 4) (27). NRG-1 is widely expressed throughout development and adulthood, with the highest expression observed in nervous tissue (8). In the central
nervous system, NRG-1 is required to induce neuronal differentiation, migration, and development (2, 25). A recent study
implicated NRG-1/ErbB4 signaling in the neuropathology of
schizophrenia (19), which is believed to be a neurodevelopmental disorder. In vitro studies indicate that NRG-1 can be a
neurotrophic or neuroprotective factor for cortical neurons,
dopaminergic neurons, cochlear sensory neurons (44), and
cereberal neurons following ischemia (36). NRG/ErbB signaling stimulates neurite outgrowth in several populations of
primary neurons, such as hippocampal neurons and cerebellar
granule cells (14, 31).
NEUREGULIN 1 (NRG-1) IS A
* J.-J. Yao and J. Sun contributed equally to this work.
Address for reprint requests and other correspondence: M. Yan-Ai, School
of Life Sciences and, Institutes of Brain Science, Fudan Univ., Shanghai
200433, PR China (e-mail: [email protected]).
http://www.ajpcell.org
Cerebellar granule neurons (CGNs) are small glutamatergic
cells and constitute the largest homogeneous neuronal population in the mammalian brain. Due to their postnatal generation
and well-defined developmental pathway in vitro, cultures of
primary CGNs have been established as a model for studying
neuronal maturation, differentiation, and synaptic plasticity (9,
39). Previous studies have indicated that growth and differentiation factors can either stimulate or inhibit CGN development
and maturation by regulating multiple signaling pathways (41,
45). In addition, NRG-1 selectively induces the expression of
the GABAA receptor ␤2-subunit in CGNs by binding to the
ErbB4 receptor and recruiting PSD-95 (40, 41).
One characteristic of CGN development and maturation is
regulation of the expression of K⫹ channels (28, 35). Primary
culture CGNs display several voltage-activated outward K⫹
currents (26). We previously reported that the enhancement of
delayed-rectifier outward (IK) and fast transient outward (IA)
potassium currents was associated with CGN apoptosis (17,
20). In addition, IK also plays a role in promoting CGN
migration and maturation (24, 45). Recently, our study indicated that neuritin, a new neurotrophic factor that is involved in
activity-dependent synaptic plasticity, activates the insulin receptor pathway to upregulate Kv4.2-mediated IA in rat cerebellar granule neurons (43), indicating that neurotrophic factors may alter the status and function of CGNs by modulation
of the expression of different potassium channels.
The goal of this study was to examine whether NRG-1
modulates K⫹ channels as part of its role in synaptic activity
and neurodevelopment. To test this hypothesis, we examined
the effect of NRG-1 on K⫹ channels in rat CGNs. We found
that NRG-1 increases IA density by regulating the expression
of Kv4.2 potassium channel ␣-subunits and that activation of
Akt/mTOR signaling by NRG-1-ErbB4 is required.
EXPERIMENTAL PROCEDURES
Chemicals. Recombinant human NRG-1 was purchased from Pepro
Tech (Rocky Hill, NJ). Triethanolamine (TEA), tetrodotoxin (TTX),
5,6-dichlorobenzimidazole 1-␤-D-ribofuranoside (DRB), cycloheximide, AG1478, rapamycin, LY294002, and poly-L-lysine were purchased from Sigma (St. Louis, MO). Fetal calf serum, DMEM, and
antibiotic-antimycotic solution were purchased from GIBCO Life
Technologies (Grand Island, NY).
Cell culture. All experimental procedures were carried out in
accordance with the European guidelines for the care and use of
laboratory animals (Council Directive 86/609/EEC). The protocol was
approved by the Committee on the Ethics of Animal Experiments of
the Fudan University. Cells were derived from the cerebella of
7-day-old Sprague-Dawley rat pups as described previously (29).
Isolated cells were plated at a density of 106 cells/ml onto 35 mm Petri
dishes coated with poly-L-lysine (1 ␮g/ml). Cultured cells were
incubated at 37°C in 5% CO2 in DMEM supplemented with 10% fetal
Licensed under Creative Commons Attribution CC-BY 3.0: the American Physiological Society. ISSN 0363-6143.
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Yao JJ, Sun J, Zhao QR, Wang CY, Mei YA. Neuregulin-1/ErbB4
signaling regulates Kv4.2-mediated transient outward K⫹ current through the
Akt/mTOR pathway. Am J Physiol Cell Physiol 305: C000–C000, 2013.
First published May 22, 2013; doi:10.1152/ajpcell.00041.2013.—
Neuregulin-1 (NRG-1) is a member of a family of neurotrophic
factors that is required for the differentiation, migration, and development of neurons. NRG-1 signaling is thought to contribute to both
neuronal development and the neuropathology of schizophrenia,
which is believed to be a neurodevelopmental disorder. However, few
studies have investigated the role of NRG-1 on voltage-gated ion
channels. In this study, we report that NRG-1 specifically increases
the density of transient outward K⫹ currents (IA) in rat cerebellar
granule neurons (CGNs) in a time-dependent manner without modifying the activation or inactivation properties of IA channels. The
increase in IA density is mediated by increased protein expression of
Kv4.2, the main ␣-subunit of the IA channel, most likely by upregulation of translation. The effect of NRG-1 on IA density and Kv4.2
expression was only significant in immature neurons. Mechanistically,
both Akt and mammalian target of rapamycin (mTOR) signaling
pathways are required for the increased NRG-1-induced IA density
and expression of Kv4.2. Moreover, pharmacological blockade of the
ErbB4 receptor reduced the effect of NRG-1 on IA density and Kv4.2
induction. Our data reveal, for the first time, that stimulation of ErbB4
signaling by NRG-1 upregulates the expression of K⫹ channel proteins via activation of the Akt/mTOR signaling pathway and plays an
important role in neuronal development and maturation. NRG1 does
not acutely change IA and delayed-rectifier outward (IK) of rat CGNs,
suggesting that it may not alter excitability of immature neurons by
altering potassium channel property.
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NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
medium alone (DMEM with free serum) as a control. The test agents
including different drugs were added to the bottom wells. The top
wells were seeded with cell suspension. Then, 106 cells/ml in 100 ␮l
serum-free DMEM were added to the top wells and incubated for 12
h. All incubations were carried out under air/CO2 95%-5%, at 37°C.
At the end of experiments, filters were washed twice with PBS, and
cells on the upper surface were removed using cotton swabs. Cells on
the lower filter surface were fixed in 4% PFA (15–20 min) and stained
with crystal violet staining solution and then examined under an
Olympus BH-2 microscope. We counted the cells in 10 random
high-power fields (⫻400), and the mean count was determined from
duplicate experiments.
RESULTS
NRG-1 increased the IA density of CGNs in a time- and
concentration-dependent manner. To determine the potential
role of NRG-1 on CGNs, we asked whether NRG-1 affected
the IA. The IA was evoked by a 200 ms depolarization to ⫹40
mV from a holding potential of ⫺100 mV in the presence of 20
mM TEA, which suppresses the IK and permits better resolution of the IA. Incubation of CGNs with NRG-1 significantly
enhanced the IA density. The data obtained from 92 neurons
showed that incubation of CGNs with 1 or 10 nM NRG-1 for
24 h increased the current density by 22.5 ⫾ 3.1% (n ⫽ 44) or
29.5 ⫾ 3.9% (n ⫽ 48), respectively (Fig. 1A). NRG-1, however, did not affect the IA amplitude of CGNs when applied
acutely to the bath solution by the perfusion system (Fig. 1B).
In the present or absence of 10 nM NRG-1, IA amplitude was
2,017.70 ⫾ 189.30 and 1,897.52 ⫾ 203.41 pA, respectively
(n ⫽ 17; P ⬎ 0.05). Moreover, NRG-1 did not increase the IK
current density (Fig. 1C). Incubation of CGNs with 10 nM
NRG-1 for 24 h lightly increased the current density by 2.7 ⫾
5.3% (from 830.47 ⫾ 40.21 to 852.90 ⫾ 40.62 pA; n ⫽ 27;
P ⬎ 0.05). To address whether the NRG-1-induced IA density
was time dependent, neurons were incubated with 10 nM
NRG-1 for different durations and the IA density was determined. After 12-, 24-, and 36-h incubation, NRG-1 was found
to augment IA density by 15.8 ⫾ 2.3% (n ⫽ 38), 28.6 ⫾ 3.6%
(n ⫽ 41), and 8.2 ⫾ 3.8% (n ⫽ 37), respectively (Fig. 1D). We
also found that the effect of NRG-1 on the induction of the IA
was dependent on the number of days that the CGNs were in
culture (Fig. 1E). Together, these data indicate that incubating
CGNs at 5 different days in culture (DIC) with 10 nM NRG-1
for 24 h produced the most significant increase in IA density.
Therefore, we selected 10 nM NRG-1 for 24 h in the subsequent series of study.
The effects of NRG-1 on the activation and inactivation
properties of IA were also explored using corresponding experimental protocols. In steady-state activation experiments,
membrane potential was held at ⫺100 mV and the IA was
evoked by a 200-ms depolarizing pulse from a first pulse
potential of ⫺60 to ⫹60 mV in 10-mV steps in 10-s intervals.
Data were analyzed using the equation GK ⫽ IK/(Vm ⫺ Vrev),
where GK is the membrane K⫹ conductance, Vm is the membrane potential, and Vrev is the reversal potential for K⫹. The
steady-state activation of the IA recorded from neurons maintained in control medium or in medium containing 10 nM
NRG-1 is presented in Fig. 2A. The IA activation curve was
obtained by plotting normalized conductance as a function of
the command potential (Fig. 2B). Incubation with 10 nM
NRG-1 for 24 h did not affect the activation curve. The current
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calf serum, insulin (5 ␮g/ml), KCl (25 mM), and 1% antibioticantimycotic solution. After culture for 24 h, cytosine ␤-D-arabinofuranoside (5 ␮M) was added to the culture medium to inhibit the
proliferation of nonneuronal cells. Cells were used for experiments
after 2–7 days in culture unless otherwise indicated.
Patch-clamp recordings. Whole-cell currents of granule neurons
were recorded using conventional patch-clamp techniques. Before IA
current recording, the culture medium was replaced with a bath
solution containing the following (in mM): 125 NaCl, 2.5 KCl, 10
HEPES, 1 MgCl2, 0.001 TTX, 20 TEA, and 10 glucose (pH adjusted
to 7.4 with NaOH). Soft-glass recording pipettes were filled with an
internal solution containing the following (in mM): 135 potassium
gluconate, 10 KCl, 10 HEPES, 1 CaCl2, 1 MgCl2, 10 EGTA, 1 ATP,
and 0.1 GTP (with pH adjusted to 7.3 using KOH). The pipette
resistance was 4 – 6 M⍀ after filling with an internal solution. The
cultured granule cells selected for electrophysiological recording
exhibited common morphological characteristics of healthy cells, such
as fusiform soma with two main neurites of similar size. In addition,
the mean capacitance of the recorded cells in the control group
(6.82 ⫾ 0.29 pF) and the neuregulin treatment group (7.13 ⫾ 0.33 pF)
showed no significant difference (P ⫽ 0.473), indicating that the
granule cells used in the experiments were comparable. All recordings
were performed at room temperature (23–25°C).
Data acquisition and analysis. All currents were recorded using an
multiclamp 200B amplifier (Axon Instruments, Foster City, CA)
operated in voltage-clamp mode. Data acquisition and analysis were
performed with pClamp 8.01 software (Axon Instruments) and/or
Origin6.1 analysis software (Microcal Software, Northampton, MA).
Quantity One software (Version 4.6.2; Bio-Rad Laboratories) was
used for background subtraction and for quantification of immunoblotting data. The results were analyzed using one-way ANOVA for
comparisons between multiple groups and using Student’s t-tests
for comparisons between two samples. The values are given as the
means ⫾ SE with n as the number of neurons for the electrophysiological recordings and calcium imaging experiments and the number
of replicates for the molecular biological experiments. For electrophysiological experiments, data were collected from at least four
different batches of neurons, thereby minimizing bias resulting from
culture conditions. A P value 0.05 was considered to be statistically
significant.
Western blot analysis. Cells were lysed in HEPES-NP40 lysis
buffer (20 mM HEPES, 150 mM NaCl, 0.5% NP-40, 10% glycerol, 2
mM EDTA, 100 ␮M Na3VO4, 50 mM NaF, pH 7.5, and 1% proteinase inhibitor cocktail) on ice for 30 min. After centrifugation, the
supernatant was mixed with 2⫻ sodium dodecyl sulfate loading buffer
and boiled for 5 min. The proteins were separated on 10% SDSpolyacrylamide gels and transferred to polyvinylidene difluoride
membranes (Millipore). The membranes were blocked with 10%
nonfat milk and incubated at 4°C overnight with a mouse monoclonal
antibody against Kv4.2 (1:2,000; University of California, Davis), a
rabbit monoclonal antibody against phosphorylated Akt (1:2,000; Cell
Signaling Technology), a rabbit polyclonal antibody against phosphorylated mTOR (1:1,000; Cell Signaling Technology), or a mouse
monoclonal antibody against GAPDH (1:10,000; KangChen BioTech). After being washed extensively in TBS-Tween, the membranes were incubated with horseradish peroxidase-conjugated antimouse or anti-rabbit IgG (1:10,000; KangChen Bio-Tech) for 2 h at
room temperature. Chemiluminescent signals were generated using a
SuperSignal West Pico trial kit (Pierce) and detected by exposure to
X-ray film or by using the ChemiDoc XRS System (Bio-Rad Laboratories).
Transwell cell migration assays. Cell culture inserts equipped with
an 8-␮m pore member of 0.3 cm2 were placed in 24-well culture
dishes, forming the upper and lower compartments of the assay,
respectively (COSTAR Corning, Cambridge, MA). Most experiments
were conducted using granule cells prepared from the cerebellum of
7-day-old Sprague-Dawley rat pups. The bottom wells contained
NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
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was half-activated at ⫺8.5 ⫾ 1.3 mV (n ⫽ 27) and ⫺7.3 ⫾ 0.8
mV (n ⫽ 25) for the control and NRG-1-treated groups,
respectively (P ⬎ 0.05). These data suggest that NRG-1
treatment does not alter the steady-state activation of the
voltage dependence of IA.
To study steady-state inactivation of IA, currents were elicited using 1 s conditioning prepulses from ⫺110 to 0 mV
before a 200-ms test pulse of ⫹50 mV. After normalizing each
current amplitude to the maximal current, the amplitude obtained from the ⫺110 mV prepulse was used as a function of
the conditioning prepulse potential and fitted to the function
IA/IA-max ⫽ 1/{1 ⫹ exp[(Vm1/2 ⫺ Vm)/k]}. Hence, we ob-
tained an inactivation curve of the IA and calculated the Vh50
(the voltage at which the current amplitude was half-inactivated). Similarly, the inactivation curve of the IA was not
significantly shifted after incubation with 10 nM NRG-1 for 24
h (Fig. 2, C and D). The half-maximal inactivation voltage for
the control and NRG-1-treated groups was ⫺73.0 ⫾ 4.7 mV
(n ⫽ 20) and ⫺76.7 ⫾ 2.4 mV (n ⫽ 22), respectively (P ⬎
0.05). These data indicate that NRG-1 does not affect IA
inactivation kinetics. Together, the electrophysiological recordings demonstrate that the NRG-1-mediated enhancement
of IA amplitude is not associated with the modification of IA
activation or inactivation properties.
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Fig. 1. Neuregulin-1 (NRG-1) enhanced transient outward K⫹ currents (IA) densities in a concentration- and time-dependent manner in cerebellar granule neurons
(CGNs). A: IA obtained from neurons maintained in control medium or medium with different concentrations of NRG-1. IA was elicited by depolarizing pulses
to ⫹40 mV from a holding potential of ⫺100 mV in the presence of 20 mM TEA. Top: representative recording samples. Bottom: statistical analysis. B: NRG-1
did not affect the IA amplitude of CGNs when applied acutely to the bath solution by the perfusion system. C: NRG-1 did not affect delayed-rectifier outward
(IK) densities in CGNs. IK currents were elicited by depolarizing pulses to ⫹50 mV from a holding potential of ⫺50 mV in the presence of 5 mM 4-aminopyridine
(4-AP). D: IA density in CGNs maintained for different incubation times in control medium or in medium containing 10 nM NRG-1. E: IA density in CGNs at
different days in culture (DIC) maintained in control medium or in medium containing 10 nM NRG-1 for 24 h. Data are shown as the means ⫾ SE. *P ⬍ 0.05,
compared with the corresponding control by the unpaired t-test.
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NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
NRG-1 upregulates the expression of Kv4.2 by promoting
translation of Kv4.2 mRNA. Next, we hypothesized that the
NRG-1-induced IA density might be due to upregulation of
potassium channel expression levels. The key ␣-subunits ac-
counting for the IA in CGNs include Kv4.2, Kv4.3, and Kv1.1
(18). Because our previous study indicated that Kv4.2 was the
main ␣-subunit that is sensitive to neurotrophic factors, we
focused our investigation on Kv4.2. Antibodies to Kv4.2,
Fig. 3. NRG-1 increased the expression levels of the Kv4.2 ␣-subunit in CGNs. A: Western blot showing the effect of 10 nM NRG-1 on different Kv channels’
subunits expression levels in CGNs. Top: representative Western blots. Bottom: statistical analysis. Data obtained from 5 independent experiments are shown
as the means ⫾ SE. B: effect of NRG-1 on Kv4.2 ␣-subunit protein expression levels in CGNs at different DIC. C: Kv4.2 ␣-subunit protein expression levels
in CGNs maintained for different incubation times in control medium or in medium containing 10 nM NRG-1. Data obtained from 5 independent experiments
are shown as the means ⫾ SE. *P ⬍ 0.05, compared with corresponding control by the unpaired t-test.
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Fig. 2. NRG-1 did not alter the steady-state
activation and inactivation properties of IA
channels. A: IA recorded using an activation
protocol in neurons maintained in control medium or medium containing 10 nM NRG-1.
B: steady-state activation curves (G/Gmax) of
IA obtained by plotting the normalized conductance as a function of command potential obtained from control and NRG-1-treated
groups. Data points were fitted using the Boltzmann function. Data points are shown as the
means ⫾ SE (n ⫽ 27 for the control group, and
n ⫽ 25 for the NRG-1-treated group). C: IA
recorded using an inactivation protocol in neurons maintained in control medium or medium
containing 10 nM NRG-1. D: steady-state inactivation curves (I/Imax) for IA were obtained
from the control and NRG-1-treated CGNs
using inactivation protocols. Data points were
fitted using the Boltzmann function. Data points
are shown as the means ⫾ SE (n ⫽ 20 for the
control group, and n ⫽ 22 for the NRG-1treated group).
NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
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Kv4.3, and Kv1.1 ␣-subunit were used to measure their protein
expression levels after incubation of CGNs with NRG-1. Western blotting indicated that protein levels of Kv4.2 were significantly enhanced following incubation of CGNs with NRG-1
for 24 h. There was, however, no significant change in the
protein expression levels of Kv4.3 and Kv1.1 (Fig. 3A; n ⫽ 5;
P ⬎ 0.05). Kinetic of Kv4.2 induction was in parallel to the
increase in IA amplitudes upon NRG-1 treatment (Fig. 3, B and
C). These data indicate that NRG-1 increases IA density by
upregulating the expression of Kv4.2.
The Kv 4.2 gene expression change in response to neuritin is
thought to occur at the transcriptional level (43). Therefore, we
used a transcription inhibitor, DRB, to test whether Kv4.2
␣-subunit mRNA levels were increased by NRG-1. However,
coincubation of CGNs with NRG-1 (10 nM) and DRB (10 ␮M)
failed to abolish the NRG-1-induced increase in IA density and
the increased expression of Kv4.2 (Fig. 4, A and B). DRB itself
reduced IA density by 34.4 ⫾ 1.7%, but incubation with
NRG-1 in the presence of DRB upregulated the IA density by
38.9 ⫾ 0.6% vs. CGNs incubated with DRB alone (P ⬍ 0.05),
an increase comparable to that observed in cells treated with
NRG-1 alone. These results suggested that ongoing transcription is not required for the NRG-1-dependent increase in IA
density and upregulation of the expression of Kv4.2.
We then addressed whether NRG-1 upregulates the levels of
Kv4.2 by promoting the translational efficiency of Kv4.2
mRNA. To test this hypothesis, CGNs were incubated with
NRG-1 in the presence or absence of the translation inhibitor
cycloheximide (10 ␮M), and the IA density and Kv4.2 expression levels were determined. As shown in Fig. 5, A and B,
incubation with cycloheximide completely abolished the effects of NRG-1 on both the IA density and the expression of
Fig. 5. Inhibition of translation abolished the
NRG-1-induced increase in the IA density
and the expression of Kv4.2. A: effects of the
translation inhibitor cycloheximide (Cyc) on
the NRG-1-induced increase in IA density.
Data are shown as the means ⫾ SE. *P ⬍
0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.
B: Western blot analysis of the effect of Cyc
on NRG-1-induced upregulation of the expression of Kv4.2. Data obtained from five
independent experiments are shown as the
means ⫾ SE. *P ⬍ 0.05, compared with the
vehicle control (medium without NRG-1) by
the unpaired t-test.
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Fig. 4. Inhibition of transcription fails to abolish the NRG-1-induced increase in the IA density and the expression of Kv4.2. A: effects of
the transcriptional inhibitor 5,6-dichlorobenzimidazole 1-␤-D-ribofuranoside (DRB), on the
NRG-1-induced increase in IA density. *P ⬍
0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.
B: effects of DRB on the NRG-1-induced upregulation of the expression of Kv4.2 measured by Western blot. Data obtained from 5
independent experiments are shown as the
means ⫾ SE. *P ⬍ 0.05, compared with the
vehicle control (medium without NRG-1) by
the unpaired t-test.
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NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
Similarly, the induction of the Kv4.2 ␣-subunit was decreased
to ⫺9 ⫾ 0.1%. We also tested the effect of AST-1306, a novel
irreversible inhibitor of the epidermal growth factor receptor 1
and 2, which has a much lower IC50 for the ErbB4 than ErbB2
receptor (42). Our study of its effects on neuregulin-induced IA
density and Kv4.2 expression, presented in Fig. 6, C and D,
indicated that in the presence of 2 nM AST1306, the IA density
and Kv4.2 ␣-subunit expression increased by NRG-1 were
significantly reduced to ⫺3.56 ⫾ 8.0% (n ⫽ 24; P ⬍ 0.05) and
⫺5.6 ⫾ 7.9% (n ⫽ 5; P ⬍ 0.05), respectively. These data
indicate that NRG-1 may activate ErbB4 receptor pathway to
increase the IA density and Kv4.2 protein level.
Phosphatidylinositol 3-kinase (PI3K)-Akt-S6K pathways
are frequently activated by NRG-1-induced stimulation of
ErbB receptor homo- or heterodimers (27). In addition to
ErbB4 activity, we also determined whether the Akt/mTOR
Fig. 6. Activation of the ErbB4 receptor is
needed for the NRG-1-induced increase in IA
density and increased Kv4.2 ␣-subunit expression in CGNs. A, AG 1478, the blocker of the
ErbB4 receptor, reduced the NRG-1-induced
increase in IA density. Data are shown as the
means ⫾ SE. *P ⬍ 0.05, compared with the
vehicle control (medium without NRG-1) by
unpaired t-test. B: Western blot analysis of the
effect of 5 ␮M AG 1478 on NRG-1-induced
upregulation of expression of Kv4.2. Data obtained from 5 independent experiments are
shown as the means ⫾ SE. *P ⬍ 0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test. C: AST1306, another blocker of the ErbB4 receptor,
reduced the NRG-1-induced increase in IA
density. Data are shown as the means ⫾ SE.
*P ⬍ 0.05, compared with the vehicle control
(medium without NRG-1) by unpaired t-test.
D: Western blot analysis of the effect of 2 nM
AST-1306 on NRG-1-induced upregulation of
expression of Kv4.2. Data obtained from 5
independent experiments are shown as the
means ⫾ SE. *P ⬍ 0.05, compared with the
vehicle control (medium without NRG-1) with
an unpaired t-test.
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Kv4.2. Together these results argue that NRG-1 acts at the
translational level to enhance Kv4.2 channel protein expression
and IA density.
NRG-1-induced increases in IA density and expression of
Kv4.2 are sensitive to inhibitors of the ErbB4 receptor and the
AKT/mTOR pathway. Previous observations suggested that
NRG-1 signals through the ErbB4 receptor (27). Next, we
asked whether blockade of the ErbB4 pathway would affect the
NRG-1-induced increase in IA density and Kv4.2 protein level.
To address this question, we used AG 1478, the EGF receptor
tyrosine kinase inhibitor that also inhibits the ErbB4 receptor
Tyr kinase. Blockade of ErbB4 activity by AG 1478 reduced
NRG-1-mediated increases in both the IA density and the
Kv4.2 protein level (Fig. 6, A and B). In the presence of 5 ␮M
AG 1478, the IA density increased by NRG-1 was significantly
reduced from 28.6 ⫾ 3.6 to ⫺6.3 ⫾ 1.7% (n ⫽ 25, P ⬍ 0.05).
NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
cells were then tested using the Transwell migration assay,
which is an in vitro model for invasive migration. In this assay,
purified granule cells are plated on one side of a porous
membrane and migration through the membrane into a second
compartment is quantified. Control medium, neuregulin, AST1306, and 4-aminopyridine (4-AP), which is a specific blocker
of IA channels, were placed in the lower compartment, and
granule cells were seeded into the top wells and allowed to
migrate toward the lower surface of the separating membrane
for 12 h. Figure 8A shows that a directional granule cell
movement across the Transwell filter under vehicle control
(medium without NRG-1) and addition of 10 nM neuregulin
significantly increased granule cell migration by 51.3 ⫾
17.04% (P ⬍ 0.05). These neuregulin-induced migration effects could be modified by the addition of AST-1306, which
decreased the number of granule cells migrating through the
filter by 40.0 ⫾ 8.35%. Moreover, this increase in migration
effects induced by neuregulin was eliminated by cotreatment
with 4-AP. Preincubation of cells with 4-AP, followed by
treatment with neuregulin, only increased cell migration by
7.07 ⫾ 6.32 or 8.09 ⫾ 12.41% (Fig. 8B).
DISCUSSION
CGNs contain two major voltage-dependent outward K⫹
currents: a delayed rectifier potassium current (IK) and a fast
transient potassium current (IA). NRG-1 treatment for 24 h
failed to modulate IK in CGNs. We thus examined the effect of
neuregulin on IA in this study. The IA has been described in
neurons from many regions of the central nervous system (16,
37). Short-term modulation of the IA density could arise from
a rapid mechanism due to changes in voltage-gating properties
Fig. 7. NRG-1-induced increase in IA density and the increased expression of the Kv4.2 ␣-subunit are dependent the Akt/mammalian target of rapamycin (mTOR)
signaling pathway. A: Western blot shows the levels of activated Akt (pAkt), activated mTOR (p-mTOR) and Kv4.2 expression induced by 10 nM NRG-1 in
the presence or absence of the mTOR inhibitor, rapamycin and the Akt inhibitor, LY294002. B–D: statistical analysis shows the effect of 20 ␮M LY294002 and
50 nM rapamycin on the levels of pAkt, p-mTOR, and Kv4.2 expression. Data obtained from 5 independent experiments are shown as the means ⫾ SE. *P ⬍
0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test. E: effect of the mTOR inhibitor rapamycin and the Akt inhibitor
LY294002 on the NRG-1-induced increase in IA density. Data are shown as the means ⫾ SE. *P ⬍ 0.05, compared with the vehicle control (medium without
NRG-1) by the unpaired t-test.
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pathway, which was reported recently to affect protein synthesis and is relevant to synaptic plasticity (15), is activated by
NRG-1. After NRG-1 treatment, the phosphorylation levels of
Akt (pAkt) and mTOR (pmTOR) were significantly increased
to 74.4 ⫾ 10.3 and 63.8 ⫾ 13.2%, respectively (n ⫽ 5) at 24
h (Fig. 7, B and C). Activation of Akt/mTOR by NRG-1 was
confirmed using pharmacological inhibitors. Blockade of Akt
activity by LY294002 or mTOR activity by rapamycin prevented the NRG-1-mediated increase in pAkt and pmTOR
levels, respectively (Fig. 7, A–C). Moreover, in the presence of
20 ␮M LY294002, the increase in the pmTOR level induced
by NRG-1 was significantly inhibited. These data indicate that
the Akt/mTOR pathway is activated by NRG-1 and NRG-1
activates mTOR via the Akt kinase.
We then determined whether the Akt/mTOR pathway is
required for the NRG-1-induced upregulation of the IA density
and Kv4.2 ␣-subunit expression. Figure 7, D and E, shows that
blockade of Akt/mTOR activity by 20 ␮M LY294002 or 50
nM rapamycin prevented the NRG-1-mediated increase in
Kv4.2 protein expression and the increased IA density. In the
presence of LY294002 or rapamycin, the increase in IA density
induced by NRG-1 was reduced (Fig. 7E) from 28.6 ⫾ 3.6 to
12.5 ⫾ 18.3 and ⫺4.1 ⫾ 8.8% (n ⫽ 4 and 5; P ⬎ 0.05).
Similarly, the expression levels of the Kv4.2 ␣-subunit protein
(Fig. 7D) were decreased to 5.2 ⫾ 13.9% (n ⫽ 5) and 0.6 ⫾
12.1% (n ⫽ 5), respectively. These data indicate that the
mTOR pathway is also required for the NRG-1-stimulated
upregulation of the IA density and increase in Kv4.2 ␣-subunit
expression.
Because our previous experiments revealed the existence of
K⫹ channel-dependent migration in granule cells (22), the
effects of neuregulin on the migration of cerebellar granule
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NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
or intracellular trafficking of the channel proteins (23, 45).
Alternatively, a long-term mechanism by upregulation of channel mRNA and protein expression could be involved (30). We
observed that NRG-1 applied acutely to the bath solution does
not affect the IA amplitude. NRG-1, however, significantly
increased the IA density without modification of IA activation
and inactivation properties, indicating that the mechanism of
action of NRG-1 involves long-term effects.
Recent studies have suggested that the Kv4 shal family
forms the major component of the IA in the central nervous
system (32). Expression of the Kv4 family was found in CGNs,
pyramidal neurons of the hippocampus and cortical neurons
(30, 34). We have shown previously that Kv4.2, Kv4.3, and
Kv1.1 are the main ␣-subunits expressed in rat CGNs (18).
Upon NRG-1 treatment, only Kv4.2 ␣-subunit mRNA and
protein levels were significantly increased. More importantly,
the kinetics of Kv4.2 induction paralleled the increase in IA
upon NRG-1 treatment. These data indicate that NRG-1 specifically increases the expression of Kv4.2. Interestingly, our
recent studies indicated that neuritin, an important neurotrophin that plays multiple roles in the process of neural development, synaptic plasticity, and synaptic maturation, could
activate the IR and ERK/mTOR signaling pathways to increase
the IA amplitude and the expression of Kv4.2 in CGNs (43).
Together, the results suggested that Kv4.2 might be the main
target of neurotrophins in developing neurons.
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Fig. 8. NRG-1 stimulates the migration of cerebellar granule cells across the
Transwell membrane. A: representative microscopic fields showing cerebellar
granule cells that have migrated across an 8-␮m pore size (*). Transwell
membrane in the absence and presence of NRG-1, AST-1306, and 4-AP. Scale
bar ⫽ 20 ␮m. B: percentage of migration neuron induced by the corresponding
treatment and control media. Results are expressed as the means ⫾ SE of 3–5
separate experiments performed in duplicate. *P ⬍ 0.05, compared with the
corresponding controls.
Previous studies using cultured CGNs showed that the subcellular redistribution of Kv4.2 from the soma to the dendrites
and synapses is induced by synapse formation. The regulation
of Kv4.2 can be further influenced by synaptic activity (33).
Indeed, the expression of Kv4.2 in CGNs was increased with
the duration of the micro explant culture period (35) and CGNs
in culture (43), in agreement with our findings showing a
concomitant increase in the IA and Kv4.2 from 3 to 5 DIC.
Notably, after NRG-1 treatment, the levels of IA and Kv4.2 in
3 DIC CGNs were similar to those in 5 DIC CGNs. Moreover,
the NRG-1-induced increase in IA density and Kv4.2 expression only occurs in 3 DIC and 5 DIC CGNs. These observations suggest that the effect of NRG-1 on Kv4.2 expression
may be developmentally regulated and associated with neuronal maturation. Our result is consistent with the findings of
Ting et al. (38), which demonstrated that NRG-1 increases the
number of excitatory synapses in GABAergic interneurons,
and this effect only occurs in developing but not mature
neurons. Meanwhile, our primary data also showed that NRG-1
promoted migrations of CGNs as measured by Transwell
analysis and that incubation of CGNs with 4-AP (a blocker of
IA) suppressed the effect of NRG-1 on CGNs migration,
suggesting that the 4-AP-sensitive IA current was connected to
neuronal migration of granule cells. Although neuronal migration is the one of the signs of differentiation and mutation of
CGNs, the mechanisms involved in the NRG-1-induced increase of IA on CGNs migration remain to be tested.
It has been reported that stimulation of NRG-1/ErbB signaling upregulates the expression of NMDA and GABAA receptors in cultured cortical GABAergic interneurons and rat CGNs
in vivo (1, 41), although Gajendran et al. (13) recently found
that the developmental expression patterns of the mRNAs
encoding the NMDA and GABAA-R is normal in mice lacking
the NRG receptors ErbB2 and ErbB4. In addition to ligandgated ion channels, reports have shown that the expression of
voltage-gated ion channels is also induced by NRG-1. In
developing chick ciliary ganglion neurons, NRG-1 regulates
the functional expression of large-conductance Ca2⫹-activated
K⫹ channels (BK) in an acute and sustained manner by the
trafficking of channel proteins (4, 6, 10). Recently, the laboratory of Li et al.(22) reported that NRG-1-ErbB4 signaling
increased the excitability of parvalbumin interneurons through
downregulation of Kv1.1 which is as a risk factor for epilepsy.
However, their data demonstrated that there was no significant
change in the expression level of Kv1.1 after NRG-1 treatment
but the level of tyrosine-phosphorylation of the Kv1.1 channel
protein in the membrane was regulated by NRG-1 signaling
(22), which is a mechanism different from NRG-1-induced
upregulation of Kv4.2. This difference may be caused by
neuregulin activation of a different signal transduction pathway
involving different neuron types, different developmental
statue, and the different animal used.
Although NRG-1 is known to contain EGF-like repeats and
mediates its effects by binding to members of the ErbB
receptor family including ErbB2, ErbB3, and ErbB4, (5, 11),
ErbB4 homodimers bind NRG-1 and are activated in the CNS.
NRG-1 may function as the ligand for ErbB4 forward signaling
(27). Recently, evidence obtained from cultured postnatal hippocampal slices showed that NRG-1-ErbB4 signaling is essential for synapse maturation and plasticity (21). Coincidentally,
previous studies in CGNs demonstrated that both ErbB3 and
NEUREGLIN-1 UPREGULATED IA AND THE EXPRESSION OF KV4.2
In summary, our studies provide insight into the mechanism
used by NRG-1 to regulate Kv4.2 subunit expression in CGNs.
These studies suggest that proteins known to play important
roles in neuronal development and maturation also participate
in NRG-induced signaling. How these signaling pathways
interact and activate downstream signaling pathways leading to
translation remains to be determined.
GRANTS
This work was supported by the National Natural Science Foundation of
China (NSFC 31070745) and Shanghai Leading Academic Discipline Project
(B111). Q.-R. Zhao was supported by National Talent Training Fund in Basic
Research of China (J1030627). The monoclonal antibody Kv4.2 was obtained
from the University of California, Davis/National Institute of Neurological
Disorders and Stroke/National Institute of Mental Health NeuroMab Facility.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.-J.Y. and Y.-A.M. conception and design of research; J.-J.Y., J.S., and Q.-R.Z. performed experiments; J.-J.Y., J.S., C.-Y.W.,
and Y.-A.M. analyzed data; J.-J.Y., J.S., and C.-Y.W. interpreted results of
experiments; J.-J.Y., J.S., and Q.-R.Z. prepared figures; Y.-A.M. drafted
manuscript; Y.-A.M. edited and revised manuscript; Y.-A.M. approved final
version of manuscript.
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