Neuregulin-1 / ErbB 4 signaling regulates Kv 4 . 2-mediated transient outward K current through the Akt / mTOR pathway

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.

neuregulin; ErbB4; Akt/mTOR; Ia; Kv4.2; CGNs NEUREGULIN 1 (NRG-1) IS A 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).
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 GABA A 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 ϩ channels (28,35).Primary culture CGNs display several voltage-activated outward K ϩ currents (26).We previously reported that the enhancement of delayed-rectifier outward (I K ) and fast transient outward (I A ) potassium currents was associated with CGN apoptosis (17,20).In addition, I K 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-mediatedI A 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 ϩ channels as part of its role in synaptic activity and neurodevelopment.To test this hypothesis, we examined the effect of NRG-1 on K ϩ channels in rat CGNs.We found that NRG-1 increases I A density by regulating the expression of Kv4.2 potassium channel ␣-subunits and that activation of Akt/mTOR signaling by NRG-1-ErbB4 is required.
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 10 6 cells/ml onto 35 mm Petri dishes coated with poly-L-lysine (1 g/ml).Cultured cells were incubated at 37°C in 5% CO 2 in DMEM supplemented with 10% fetal 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 I A current recording, the culture medium was replaced with a bath solution containing the following (in mM): 125 NaCl, 2.5 KCl, 10 HEPES, 1 MgCl 2, 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 CaCl 2, 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 Ϯ 0.29 pF) and the neuregulin treatment group (7.13 Ϯ 0.33 pF) showed no significant difference (P ϭ 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 Ϯ 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.
Transwell cell migration assays.Cell culture inserts equipped with an 8-m pore member of 0.3 cm 2 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 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, 10 6 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 (ϫ400), and the mean count was determined from duplicate experiments.

NRG-1 increased the I A 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 I A .The I A was evoked by a 200 ms depolarization to ϩ40 mV from a holding potential of Ϫ100 mV in the presence of 20 mM TEA, which suppresses the I K and permits better resolution of the I A .Incubation of CGNs with NRG-1 significantly enhanced the I A 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 Ϯ 3.1% (n ϭ 44) or 29.5 Ϯ 3.9% (n ϭ 48), respectively (Fig. 1A).NRG-1, however, did not affect the I A 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, I A amplitude was 2,017.70Ϯ 189.30 and 1,897.52Ϯ 203.41 pA, respectively (n ϭ 17; P Ͼ 0.05).Moreover, NRG-1 did not increase the I K current density (Fig. 1C).Incubation of CGNs with 10 nM NRG-1 for 24 h lightly increased the current density by 2.7 Ϯ 5.3% (from 830.47 Ϯ 40.21 to 852.90 Ϯ 40.62 pA; n ϭ 27; P Ͼ 0.05).To address whether the NRG-1-induced I A density was time dependent, neurons were incubated with 10 nM NRG-1 for different durations and the I A density was determined.After 12-, 24-, and 36-h incubation, NRG-1 was found to augment I A density by 15.8 Ϯ 2.3% (n ϭ 38), 28.6 Ϯ 3.6% (n ϭ 41), and 8.2 Ϯ 3.8% (n ϭ 37), respectively (Fig. 1D).We also found that the effect of NRG-1 on the induction of the I A 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 I A 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 I A were also explored using corresponding experimental protocols.In steady-state activation experiments, membrane potential was held at Ϫ100 mV and the I A was evoked by a 200-ms depolarizing pulse from a first pulse potential of Ϫ60 to ϩ60 mV in 10-mV steps in 10-s intervals.Data were analyzed using the equation where G K is the membrane K ϩ conductance, V m is the membrane potential, and V rev is the reversal potential for K ϩ .The steady-state activation of the I A recorded from neurons maintained in control medium or in medium containing 10 nM NRG-1 is presented in Fig. 2A.The I A 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 was half-activated at Ϫ8.5 Ϯ 1.3 mV (n ϭ 27) and Ϫ7.3 Ϯ 0.8 mV (n ϭ 25) for the control and NRG-1-treated groups, respectively (P Ͼ 0.05).These data suggest that NRG-1 treatment does not alter the steady-state activation of the voltage dependence of I A .
To study steady-state inactivation of I A , currents were elicited using 1 s conditioning prepulses from Ϫ110 to 0 mV before a 200-ms test pulse of ϩ50 mV.After normalizing each current amplitude to the maximal current, the amplitude obtained from the Ϫ110 mV prepulse was used as a function of the conditioning prepulse potential and fitted to the function Hence, we ob-tained an inactivation curve of the I A and calculated the V h50 (the voltage at which the current amplitude was half-inactivated).Similarly, the inactivation curve of the I A 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 Ϫ73.0 Ϯ 4.7 mV (n ϭ 20) and Ϫ76.7 Ϯ 2.4 mV (n ϭ 22), respectively (P Ͼ 0.05).These data indicate that NRG-1 does not affect I A inactivation kinetics.Together, the electrophysiological recordings demonstrate that the NRG-1-mediated enhancement of I A amplitude is not associated with the modification of I A activation or inactivation properties.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 ϭ 5; P Ͼ 0.05).Kinetic of Kv4.2 induction was in parallel to the increase in I A amplitudes upon NRG-1 treatment (Fig. 3, B and  C).These data indicate that NRG-1 increases I A 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 I A density and the increased expression of Kv4.2 (Fig. 4, A and B).DRB itself reduced I A density by 34.4 Ϯ 1.7%, but incubation with NRG-1 in the presence of DRB upregulated the I A density by 38.9 Ϯ 0.6% vs. CGNs incubated with DRB alone (P Ͻ 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 I A 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 I A 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 I A density and the expression of    (27).Next, we asked whether blockade of the ErbB4 pathway would affect the NRG-1-induced increase in I A 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 I A density and the Kv4.2 protein level (Fig. 6, A and B).In the presence of 5 M AG 1478, the I A density increased by NRG-1 was significantly reduced from 28.6 Ϯ 3.6 to Ϫ6.3 Ϯ 1.7% (n ϭ 25, P Ͻ 0.05).
Similarly, the induction of the Kv4.2 ␣-subunit was decreased to Ϫ9 Ϯ 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 IC 50 for the ErbB4 than ErbB2 receptor (42).Our study of its effects on neuregulin-induced I A density and Kv4.2 expression, presented in Fig. 6, C and D, indicated that in the presence of 2 nM AST1306, the I A density and Kv4.2 ␣-subunit expression increased by NRG-1 were significantly reduced to Ϫ3.56 Ϯ 8.0% (n ϭ 24; P Ͻ 0.05) and Ϫ5.6 Ϯ 7.9% (n ϭ 5; P Ͻ 0.05), respectively.These data indicate that NRG-1 may activate ErbB4 receptor pathway to increase the I A 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  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 Ϯ 10.3 and 63.8 Ϯ 13.2%, respectively (n ϭ 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 I A 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 I A density.In the presence of LY294002 or rapamycin, the increase in I A density induced by NRG-1 was reduced (Fig. 7E) from 28.6 Ϯ 3.6 to 12.5 Ϯ 18.3 and Ϫ4.1 Ϯ 8.8% (n ϭ 4 and 5; P Ͼ 0.05).Similarly, the expression levels of the Kv4.2 ␣-subunit protein (Fig. 7D) were decreased to 5.2 Ϯ 13.9% (n ϭ 5) and 0.6 Ϯ 12.1% (n ϭ 5), respectively.These data indicate that the mTOR pathway is also required for the NRG-1-stimulated upregulation of the I A density and increase in Kv4.2 ␣-subunit expression.
Because our previous experiments revealed the existence of K ϩ channel-dependent migration in granule cells (22), the effects of neuregulin on the migration of cerebellar granule 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, AST-1306, and 4-aminopyridine (4-AP), which is a specific blocker of I A 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 Ϯ 17.04% (P Ͻ 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 Ϯ 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 Ϯ 6.32 or 8.09 Ϯ 12.41% (Fig. 8B).

DISCUSSION
CGNs contain two major voltage-dependent outward K ϩ currents: a delayed rectifier potassium current (I K ) and a fast transient potassium current (I A ). NRG-1 treatment for 24 h failed to modulate I K in CGNs.We thus examined the effect of neuregulin on I A in this study.The I A has been described in neurons from many regions of the central nervous system (16,37).Short-term modulation of the I A density could arise from a rapid mechanism due to changes in voltage-gating properties  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 I A amplitude.NRG-1, however, significantly increased the I A density without modification of I A 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 I A 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 I A 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 I A 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.
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 I A and Kv4.2 from 3 to 5 DIC.Notably, after NRG-1 treatment, the levels of I A and Kv4.2 in 3 DIC CGNs were similar to those in 5 DIC CGNs.Moreover, the NRG-1-induced increase in I A 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 I A ) suppressed the effect of NRG-1 on CGNs migration, suggesting that the 4-AP-sensitive I A 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 I A on CGNs migration remain to be tested.
It has been reported that stimulation of NRG-1/ErbB signaling upregulates the expression of NMDA and GABA A 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 GABA A -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 Ca 2ϩ -activated K ϩ 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

Fig. 1 .
Fig. 1.Neuregulin-1 (NRG-1) enhanced transient outward K ϩ 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 ϩ40 mV from a holding potential of Ϫ100 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 ϩ50 mV from a holding potential of Ϫ50 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 Ϯ SE. *P Ͻ 0.05, compared with the corresponding control by the unpaired t-test.

C199NEUREGLIN- 1 UPREGULATEDFig. 2 .
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 Ϯ SE (n ϭ 27 for the control group, and n ϭ 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 Ϯ SE (n ϭ 20 for the control group, and n ϭ 22 for the NRG-1treated group).

Fig. 3 .
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 Ϯ 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 Ϯ SE. *P Ͻ 0.05, compared with corresponding control by the unpaired t-test.

Fig. 4 .
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 Ͻ 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 Ϯ SE. *P Ͻ 0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.

Fig. 5 .
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 Ϯ SE. *P Ͻ 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 Ϯ SE. *P Ͻ 0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.

C201NEUREGLIN- 1 UPREGULATED
IA AND THE EXPRESSION OF KV4.2 AJP-Cell Physiol • doi:10.1152/ajpcell.00041.2013• www.ajpcell.orgKv4.2.Together these results argue that NRG-1 acts at the translational level to enhance Kv4.2 channel protein expression and I A density.NRG-1-induced increases in I A 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

Fig. 6 .
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 Ϯ SE. *P Ͻ 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 Ϯ SE. *P Ͻ 0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.C: AST-1306, another blocker of the ErbB4 receptor, reduced the NRG-1-induced increase in IA density.Data are shown as the means Ϯ SE. *P Ͻ 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 Ϯ SE. *P Ͻ 0.05, compared with the vehicle control (medium without NRG-1) with an unpaired t-test.

Fig. 7 .
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 Ϯ SE. *P Ͻ 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 Ϯ SE. *P Ͻ 0.05, compared with the vehicle control (medium without NRG-1) by the unpaired t-test.

Fig. 8 .
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 ϭ 20 m.B: percentage of migration neuron induced by the corresponding treatment and control media.Results are expressed as the means Ϯ SE of 3-5 separate experiments performed in duplicate.*P Ͻ 0.05, compared with the corresponding controls.