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. Author manuscript; available in PMC: 2021 Jun 23.
Published in final edited form as: Neurosci Lett. 2014 Sep 8;583:142–147. doi: 10.1016/j.neulet.2014.08.051

Potassium channel Kv2.1 is regulated through protein phosphatase-1 in response to increases in synaptic activity

Benjamin Siddoway a,*,1, Hailong Hou a,1, Jinnan Yang b, Lu Sun a, Hongtian Yang a, Guo-yong Wang b, Houhui Xia a
PMCID: PMC8221410  NIHMSID: NIHMS1715005  PMID: 25220706

Abstract

The functional stability of neurons in the face of large variations in both activity and efficacy of synaptic connections suggests that neurons possess intrinsic negative feedback mechanisms to balance and tune excitability. While NMDA receptors have been established to play an important role in glutamate receptor-dependent plasticity through protein dephosphorylation, the effects of synaptic activation on intrinsic excitability are less well characterized. We show that increases in synaptic activity result in dephosphorylation of the potassium channel subunit Kv2.1. This dephosphorylation is induced through NMDA receptors and is executed through protein phosphatase-1 (PP1), an enzyme previously established to play a key role in regulating ligand gated ion channels in synaptic plasticity. Dephosphorylation of Kv2.1 by PP1 in response to synaptic activity results in substantial shifts in the inactivation curve of IK, resulting in a reduction in intrinsic excitability, facilitating negative feedback to neuronal excitability.

Keywords: KV2.1, NMDA receptor, Synaptic activity, Dephosphorylation, PP1

1. Introduction

Neurons contain high numbers of individual excitatory inputs. The relative stability amongst thousands of dynamic inputs in billions of neurons in the brain suggests that neurons intrinsically manage excitability through a number of mechanisms [1]. Multiple intracellular and intercellular feedback mechanisms have been proposed for this level of network regulation. One well known example is that of homeostatic plasticity, wherein efficacy of synaptic connections are globally reduced in response to prolonged periods of increased activity [2]. These modifications at ligand-gated ion channels generally require prolonged induction times and do not appear to occur on a time scale relevant to short-term maintenance of network stability. On the other hand, acute, dynamic regulation of voltage gated ion channels is a theoretically attractive candidate for short-term negative feedback [1,3,4].

Potassium channels containing the subunit Kv2.1 are the primary source for the delayed rectifier potassium current in primary neurons [5]. Similar to ligand gated ion channels such as AMPA receptors, the activation and inactivation kinetics of this channel can be regulated by the phosphorylation state of a number of cytosolic amino acids [4,6–12,30,31]. Significant progress has been made in establishing Kv2.1 as an important component in neuronal response to ischemic/excitotoxic conditions, as modeled through bath glutamate application [4]. However, how these channels may contribute to the regulation of excitability through negative feedback in non-pathological conditions has not been fully elucidated.

The enzyme protein phosphatase-1 (PP1) is highly expressed in primary neurons [13–15]. PP1 has previously been shown to regulate ligand gated ion channels such as AMPA receptors in response to synaptic depolarization. For example, PP1 has been shown to dephosphorylate the GluA1 subunit C-terminus at S845, and dephosphorylation at this site directly shifts the open channel probability [16,17]. PP1-mediated dephosphorylation has been shown to be increased through synaptic events [25] as well as directly through NMDA receptor activity [26], however, the role of PP1 in the regulation of intrinsic excitability in primary neurons has not been fully assessed.

In this study, we investigate how Kv2.1 is modulated in response to increases in synaptic activity. We show that Kv2.1 is dephosphorylated by protein phosphatase-1 and this dephosphorylation is initiated through NMDA receptors. Direct investigation into the effect of increased activity on delayed rectifier potassium (IK) current revealed a reduction of the inactivation kinetics of this current, resulting in a hyperpolarizing shift, and this shift was blocked by inhibiting PP1. These results appear to demonstrate that synaptic NMDA receptors modulate intrinsic excitability through PP1 as an intracellular negative feedback mechanism that serves to modulate intrinsic excitability of neurons to compensate in situations of heightened activity.

2. Methods and materials

2.1. Primary neuronal cell cultures

Primary neurons were prepared from El8 SD rat embryos (cortex/hippocampus). Cells were plated at 0.5 × 106 per well in six well plates (~50K/cm2 for biochemistry, 100K/cm2 for electrophysiology) on poly-L-lysine (50 μg/ml in borate buffer) coated 6 well culture dishes for biochemistry (or glass coverslips for electrophysiology) in neurobasal medium supplemented with 2% B27 and 1% glutamax (Gibco), with 1 ml fresh medium per well added at DIV 2, 5 and 12, respectively. 3–4 week old neurons were used in all experiments. All experimental protocols for live animals were approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center.

2.2. Antibodies

Antibodies were obtained from NeuroMab (Kv2.1, MAb, Mouse).

2.3. Chemicals

Drugs and chemicals were purchased from Tocris Biosciences (TTX, MK801, NMDA, FK506, cyclosporine, fostrecin, 4-AP), and Sigma-Fluka (bicuculline).

2.4. Constructs, transfection and recombinant viruses

PP1 (WT, rat) was cloned into pEGFP-C1 (Clontech) and sequencing verified. PP1 or GFP alone was cloned into improved Sindbis viral expression vector of pSinRep5 (nsP2S726) [27]. Sindbis viruses were prepared as before [40]. Briefly, linearized DNA templates were transcribed using an in vitro transcription kit (Ambion) and then electroporated with helper RNA constructs into BHK cells. 24–36 h post-electroporation, virus in the supernatant was concentrated via ultracentrifuge. Infection of cortical neurons was performed by directly adding virus into growth medium and was validated at >80% infection rate.

2.5. Data analysis

For relative Kv2.1 phosphorylation, western films were scanned, color data was removed, and the average pixel intensity was acquired in the region of interest indicating dephosphorylation (line 2 in Fig. 1). Average pixel intensity in the region of interest was then normalized to the pixel intensity of the entire lane (total Kv2.1). Region of interest for relative dephosphorylation was defined as an area the width of the gel lane and 50% above and 50% below line 2 in Fig. 1, such that the total area for “relative dephosphorylation" was equal to the area of the Kv2.1 band in non-shifted, untreated controls. Identical area sizes were used for statistical comparisons across samples. The western data were statistically analyzed in accordance with previous publications that similarly analyze phosphorylation from primary neuronal cultures across experiments [28], namely, two way ANOVA was performed with a multiple comparisons test (Tukey). In electrophysiology data, two way ANOVA and a multiple comparisons test (Tukey) were performed and p values are listed in Fig. 3.

Fig. 1.

Fig. 1.

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Protein phosphatase-1 dephosphorylates Kv2.1 in response to synaptic activity. (A) Neurons were pretreated with 1 μM FK506 + 20 μM cyclosporinA (CSA) or 1 μM FK506 + 20 μM CSA + 500 nM okadaic acid for 20 min and 100 μM NMDA was added for 10 min. (B) PP1 was recombinantly expressed in neurons, 100 μM NMDA was added for 10 min to determine if PP1 expression could occlude Kv2.1 dephosphorylation induced under pathological conditions. (C) In order to determine if PP1 expression could occlude Kv2.1 dephosphorylation induced by synaptic activity, PP1 was expressed in neurons and 40 μM bicuculline and 500 μM 4-AP was added for 2 h. (D) To induce dephosphorylation of Kv2.1, 40 μM bicuculline or 40 μM bicuculline and 500 μM 4-AP was added to neurons for 2 h, neurons were pretreated for 20 min with 2 μM TTX in order to demonstrate that dephosphorylation is dependent on neuronal firing. (E) Quantification. Data in graph are expressed as means ± SEM. *p <.01. (F) To observe the time frame in which Kv2.1 was dephosphorylated, 40 μM bicuculline and 500 μM 4-AP was added to neurons at the times indicated.

Fig. 3.

Fig. 3.

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Synaptic activity shifts inactivation of Kv2.1 mediated delayed rectifier current. To determine if dephosphorylation of Kv2.1 results in a shift in channel kinetics, neurons were examined under [no treatment, 40 μM bicuculline, 500nM okadaic acid, or pretreatment with 500 nM okadaic acid for 20 min+40 μM bicuculline] for 1 h and inactivation kinetics were recorded and plotted. (A) Representative whole cell current recordings under different conditions. (B) Plotted inactivation curve of IK after different stimulations. (C) Plotted p values under different conditions, x-axis represents membrane potential from (B), comparisons where p < .05 are highlighted in green. Data in graph are expressed as means ± SEM.

2.6. Electrophysiology: voltage clamp

Outward potassium currents were recorded from E18 dissociated cultured neurons (from SD rat) in whole-cell mode. Patch pipettes with a tip resistance between 3 and 7 MΩ were pulled from thick-walled 1.5 mm-OD borosilicate glass on a Sutter Instruments puller (P-97). Whole-cell patch-clamp recordings were made with a MultiClamp 700B patch-clamp amplifier at room temperature (23–25°C). High-resistance seals were obtained by moving the patch electrode onto the cell membrane and applying gentle suction. After formation of a high-resistance seal between the electrode and the cell membrane, transient currents caused by pipette capacitance were electronically compensated by the circuit of the MultiClamp 700B patch-clamp amplifier. Recordings from cells with a seal resistance of <1 GΩ were discarded. The extracellular buffer contained (in mM) 140 NaCl, 5KC1, 2 CaC12, 1 MgC12, 10 HEPES, and 10 glucose, pH 7.3. The pipette solution contained (inmM) 140 KC1, 5 NaCl, lCaC12, 2 MgC12, 5 EGTA and 10 HEPES, pH 7.3. osmolarity, 290 mOsm. 5 mM QX-314 was included in the electrode solution to eliminate sodium currents in the recorded cell.

3. Results

Kv2.1 is phosphorylated at numerous intracellular residues and the relative phosphorylation of these sites can be measured by substantial shifts in the electrophoretic mobility of the channel in SDS-PAGE/immunoblot. Measuring shifts due to the phosphorylation state at multiple sites is well established for this molecule through numerous prior publications [4,9,11]. In Fig. 1, the three lines drawn [1–3] represent roughly three major phosphorylation states of Kv2.1; and we show that protein phosphatase-1 (PP1) contributes substantially to Kv2.1 dephosphorylation. In order to evaluate the contribution of PP1 to Kv2.1 dephosphorylation, we first used bath NMDA to induce robust pathological dephosphorylation as previously investigated [4] to line 3 in (Fig. 1A). This level of dephosphorylation is consistent with a previous study demonstrating that Kv2.1 is robustly dephosphorylated by phosphatases in response to bath glutamate stimulation. However, after 30 min pretreatment with inhibitors of PP2B (FK506 and cyclosporinA (CSA)), the level of dephosphorylation is attenuated and is not completely blocked (line 2) after NMDA treatment, as would be expected if PP2B was the sole phosphatase responsible for regulating Kv2.1. We found that pretreatment with inhibitors of both PP1 and PP2B (FK506/CSA + OA, Fig. 1A) is necessary to completely block NMDA induced dephosphorylation. These results indicate that both PP1 and PP2B contribute to NMDA induced dephosphorylation at specific residues of Kv2.1, and further verify that the electrophoretic mobility shift observed is due to changes in the phosphorylation state of Kv2.1, as phosphatase inhibitors can block this phenomenon. In order to verify the extent to which PP1 regulates Kv2.1 phosphorylation, we expressed PP1 in neurons. Expression of recombinant PP1 in neurons results in dephosphorylation of Kv2.1 to a similar level as NMDA treatment with PP2B inhibitors (line 2, Fig. 1B).

We next investigated the effect of indirectly increasing neuronal firing and synaptic activity on Kv2.1 phosphorylation. Bicuculline is a GABAA receptor antagonist and is utilized in many studies in combination with the potassium channel blocker 4-AP to induce a robust increase in neuronal firing and synaptic activity in dissociated culture preparations [18,19]. To this end, synaptic stimlation via bicuculline or bicuculline/4-AP has been demonstrated by multiple independent groups to predominantly be beneficial to neuronal viability; converse to the neurodegenerative conditions modeled via bath glutamate/NMDA induced extra-synaptic NMDA receptor stimulation and neuronal apoptosis [21,22]. Treatment with bicuculline/4-AP to disinhibit neurons and induce a physiological increase in synaptic activity results in dephosphorylation of Kv2.1 to levels which appear to (line2) be observed from both expression of PP1 and NMDA treatment in the presence of PP2B inhibitors (Fig. 1A–C). Expression of PP1 both mimics dephosphorylation observed after synaptic stimulation and occludes further bicuculline/4-AP mediated dephosphorylation (Fig. 1C), demonstrating that PP1 activity is central in activity-induced Kv2.1 dephosphorylation. Moreover, it appears that activity-induced dephosphorylation of Kv2.1 by PP1 is different from dephosphorylation observed in response to ischemic/pathological models induced via bath glutamate [4] or bath NMDA (Fig. 1A and B) stimulation.

Activity-induced dephosphorylation of Kv2.1 can be acutely induced, and is robust and persistent to at least 4 h (Fig. 1F). Treatment with bicuculline or bicuculline and 4-aminopropidine (4-AP) both result in the same level of dephosphorylation of Kv2.1. Activity-induced dephosphorylation of Kv2.1 is blocked by pretreatment with TTX (Fig. 1D and E), indicating that activity-induced dephosphorylation is dependent upon increases in neuronal firing. In order to confirm that PP1 is dephosphorylating Kv2.1 in response to increases in activity, we pretreated neurons with 500 nM okadaic acid, a PP1 inhibitor. Pretreatment with okadaic acid for 20 min blocked activity-induced dephosphorylation (Fig. 2A, B and E). Importantly, the concentration of okadaic acid used in this experiment is well established to not display any inhibition of PP2B [20], the phosphatase examined in detail in the dephosphorylation of Kv2.1 induced via pathological conditions as modeled through bath glutamate application. Further PP1 specificity for Kv2.1 was confirmed by pretreating neurons with a specific PP2A inhibitor, fostrecin, and we observed no effect from pretreatment with fostrecin (Fig. 2B).

Fig. 2.

Fig. 2.

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Synaptic activity-induced Kv2.1 dephosphorylation is mediated through PP1 and NMDA receptors. (A) Neurons were pretreated with 2 μMTTX or 500 nM okadaic acid for 20 min and 40 μM bicuculline and 500 μ 4-AP was added for 2 h. (B) Neurons were pretreated with 500 nM okadaic acid or 1 μM fostrecin for 20 min and then 40 μM bicuculline for 2 h, note: okadaic acid and fostrecin blots are from the same membrane and exposure as other lanes, cropping was performed to eliminate non-essential data. (C) Neurons were pretreated with 40 μM CNQX and 50 μM D-AP5 for 20 min and then with 40 μM bicuculline and 500 μM 4-AP for 2 h. (D) To determine the source of calcium influx that induces Kv2.1 dephosphorylation, neurons were pretreated with 75 μM MK801 for 20 min and then 40 μM Bic + 500 μM 4-AP (±), blots are displayed as run on gel. (E) Quantification. Data in graph are expressed as means±SEM. *p <.002. **p <.001.

Next, we determined upstream signaling involved in activity-induced dephosphorylation of Kv2.1 channels. Pretreatment of CNQX and D-AP5 blocks Kv2.1 dephosphorylation, further demonstrating that activity is necessary for upstream signaling (Fig. 2C). Furthermore, pretreatment with MK801, a NMDA receptor open channel blocker, significantly attenuated dephosphorylation of Kv2.1, directly implicating synaptic activity-induced NMDA receptor-dependent signaling in the regulation of Kv2.1 (Fig. 2D and E).

Kv2.1 is the predominant contributor to the delayed rectifier current in primary neurons. Previous investigations have determined that dephosphorylation of Kv2.1 under pathological or ischemic conditions results in hyperpolarizing shifts in the gating parameters of delayed rectifier potassium channel current (IK). We asked if gating parameters of IK could be regulated by indirect increases in neuronal firing and synaptic activity-induced via the GABAA receptor antagonist bicuculline. Modeled after experiments in Mohopatra et al. [8], in order to isolate IK, neurons were held at −80 mV and were subjected to a conditioning step to −10 mV (30 ms) to isolate IK and eliminate a substantial amount of fast-activating and inactivating component of IA. Neurons were re-polarized, and test pulses were delivered to evoke IK currents. Interestingly, we observed that synaptic activity induced significant shifts in the inactivation curve of IK (Fig. 3). We next observed that activity-induced hyperpolarizing shifts in inactivation parameters were blocked by 20 min pretreatment of neurons with okadaic acid (Fig. 3), which blocks activity-induced Kv2.1 dephosphorylation. Thus it appears that bicuculline treatment induced shifts IK toward hyperpolarizing conditions through dephosphorylation of Kv2.1 by PP1.

4. Discussion

Our results indicate that synaptic activity drives a response through Kv2.1 by PP1 signaling, and represents a simple, direct negative feedback loop to maintain and tune network stability in the face of robust neuronal firing and synaptic activation. Our results are interesting, considering that previous reports have documented the effects of PP2B mediated dephosphorylation on the channel [4,6]. These previous reports have indicated that PP1 may regulate Kv2.1 under basal conditions. However, our report represents a novel mechanism regarding how acute elevations in synaptic activity induce PP1-mediated regulation of this ion channel.

A previous investigation may shed some light into which cytosolic residues of Kv2.1 may be dephosphorylated by PP1 [6]. Park et al. note that the phosphorylation state of residues on the cytosolic N-terminus of Kv2.1 shift inactivation kinetics of the channel. These results may suggest that residues modulated by bicuculline induced synaptic activity lie on the N-terminus, including serine 11, phosphorylation of which has been shown to affect inactivation kinetics with limited effect on activation. Further investigations are needed to test how the N-terminus of Kv2.1 is involved in negative feedback processes.

Furthermore, we demonstrate that NMDA receptor channel opening appears to be required for activity-induced Kv2.1 dephosphorylation. Indeed, as a previous publication has also found that PP2B-mediated Kv2.1 dephosphorylation is controlled through NMDA receptors [4,10,11], it appears that almost all dephosphorylation of this channel in neurons may be governed through NMDA receptor signaling. These results are surprising, but not unprecedented; as many reports have identified that a number of divergent signaling pathways can originate through NMDA receptor signaling [21,24].

It will be interesting to determine if this negative feedback response plays a role in the induction of different forms of plasticity, particularly glutamatergic synaptic plasticity previously shown to be dependent on NMDA receptors. Future investigations are also essential to determine how this short-term plasticity of intrinsic excitability may play a role in network-wide electrophysiological stability in the brain. Of particular interest may be investigations into disorders that are characterized by network instability, such as epilepsy.

HIGHLIGHTS.

  • Protein phosphatase-1 dephosphorylates potassium channel subunit Kv2.1.

  • Synaptic activation and NMDA receptor opening induces dephosphorylation of Kv2.1.

  • Dephosphorylation of Kv2.1 induced by synaptic activity results in hyperpolarizing shifts in IK.

Acknowledgements

This work supported by NIHR01 (NS060879), NSF (IOS-0824393) and LSUHSC Research Enhancement Fund (REF) to HX.

References

  • [1].Turrigiano G, Too many cooks? Intrinsic and synaptic homeostatic mechanisms in cortical circuit refinement, Annu. Rev. Neurosci 34 (2011) 89–103. [DOI] [PubMed] [Google Scholar]
  • [2].Pozo K, Goda Y, Unraveling mechanisms of homeostatic synaptic plasticity, Neuron 66 (3)(2010)337–351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Turrigiano G, Homeostatic synaptic plasticity: local and global mechanismsfor stabilizing neuronal function, Cold Spring Harb. Perspect. Biol 4 (1) (2012) a005736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Misonou H, Mohapatra DP, Park EW, Leung V, Zhen D, Misonou K, Anderson AE, Trimmer JS, Regulation of ion channel localization and phosphorylation by neuronal activity, Nat. Neurosci 7 (7) (2004) 711–718. [DOI] [PubMed] [Google Scholar]
  • [5].Murakoshi H, Trimmer JS, Identification of the Kv2.1 K+ channel as a majorcomponent of the delayed rectifier K+ current in rat hippocampal neurons, J.Neurosci 19 (5) (1999) 1728–1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Park KS, Mohapatra DP, Misonou H, Trimmer JS, Graded regulation of theKv2.1 potassium channel by variable phosphorylation, Science 313 (5789)(2006) 976–979. [DOI] [PubMed] [Google Scholar]
  • [7].Murakoshi H, Shi G, Scannevin RH, Trimmer JS, Phosphorylation of the Kv2.1K+ channel alters voltage-dependent activation, Mol. Pharmacol 52 (5) (1997)821–828. [DOI] [PubMed] [Google Scholar]
  • [8].Mohapatra DP, Misonou H, Pan SJ, Held JE, Surmeier DJ, Trimmer JS, Regulation of intrinsic excitability in hippocampal neurons by activity-dependentmodulation of the KV2.1 potassium channel, Channels (Austin) 3 (1) (2009)46–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Misonou H, Menegola M, Mohapatra DP, Guy LK, Park KS, Trimmer JS,Bidirectional activity-dependent regulation of neuronal ion channel phosphorylation, J. Neurosci 26 (52) (2006) 13505–13514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Misonou H, Thompson SM, Cai X, Dynamic regulation of the Kv2.1 voltage-gated potassium channel during brain ischemia through neuroglial interaction,J. Neurosci 28 (34) (2008) 8529–8538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Cerda O, Trimmer JS, Activity-dependent phosphorylation of neuronal Kv2.1potassium channels by CDK5, J. Biol. Chem 286 (33) (2011) 28738–28748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Shi G, Kleinklaus AK, Marrion NV, Trimmer JS, Properties of Kv2.1 K+ channels expressed in transfected mammalian cells, J. Biol. Chem 269 (37) (1994)23204–23211. [PubMed] [Google Scholar]
  • [13].da Cruz e Silva EF, Fox CA, Ouimet CC, Gustafson E, Watson SJ, Greengard P,Differential expression of protein phosphatase-1 isoforms in mammalian brain,J. Neurosci 15 (5 Pt 1) (1995) 3375–3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF,Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG,Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R,Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halver-son KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT,Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL,Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI,Sodt AJ, Stewart NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC,Youngstrom BL, Yuan XF, Zhang B, Zwingman TA, Jones AR, Genome-wideatlas of gene expression in the adult mouse brain, Nature 445 (7124) (2007)168–176. [DOI] [PubMed] [Google Scholar]
  • [15].Allen Mouse Brain Atlas [Internet], Allen Institute for Brain Science, Seattle,WA, 2009. [Google Scholar]
  • [16].Lee HK, Barbarosie M, Kameyama K, Bear MF, Huganir RL, Regulation ofdistinct AMPA receptor phosphorylation sites during bidirectional synapticplasticity, Nature 405 (6789) (2000) 955–959. [DOI] [PubMed] [Google Scholar]
  • [17].Banke TG, Bowie D, Lee H, Huganir RL, Schousboe A, Traynelis SF, Control ofGluR1 AMPA receptor function by cAMP-dependent protein kinase, J. Neurosci 20 (1) (2000) 89–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB, Activity-dependent scaling of quantal amplitude in neocortical neurons, Nature 391(6670) (1998) 892–896. [DOI] [PubMed] [Google Scholar]
  • [19].Seeburg DP, Feliu-Mojer M, Gaiottino J, Pak DT, Sheng M, Critical role ofCDK5 and Polo-like kinase 2 in homeostatic synaptic plasticity during elevatedactivity, Neuron 58 (4) (2008) 571–583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Bialojan C, Takai A, Inhibitory effect of a marine-sponge toxin, okadaic acid,on protein phosphatases. Specificity and kinetics, Biochem. J 256 (1) (1988)283–290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Hardingham GE, Bading H, Synaptic versus extrasynaptic NMDA receptorsignalling: implications for neurodegenerative disorders, Nat. Rev. Neurosci 11 (10) (2010) 682–696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Nataraj K, Le Roux N, Nahmani M, Lefort S, Turrigiano G, Visual deprivationsuppresses L5 pyramidal neuron excitability by preventing the induction ofintrinsic plasticity, Neuron 68 (4) (2010) 750–762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Papouin T, Ladepeche L, Ruel J, Sacchi S, Labasque M, Hanini M, Groc L, Pollegioni L, Mothet JP, Oliet SH, Synaptic and extrasynaptic NMDA receptorsare gated by different endogenous coagonists, Cell 150 (3) (2012) 633–646. [DOI] [PubMed] [Google Scholar]
  • [25].Siddoway B, Altimimi H, Hou H, Petralia R, Xu B, Stellwagen D, Xia H, Anessential role for inhibitor-2 regulation of protein phosphatase-1 in synapticscaling? J. Neurosci 33 (27) (2013) 11206–11211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Hou H, Sun L, Siddoway B, Petralia R, Gu H, Yang H, Nairn A, Xia H, SynapticNMDA receptor stimulation activates PP1 by inhibiting its phosphorylation byCDK5, J. Cell Biol 203 (3) (2013) 521–535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Kim J, Dittgen T, Nimmerjahn A, Waters J, Pawlak V, Helmchen F, Schlesinger S, Seeburg PH, Osten P, Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons, J. Neurosci. Methods 133 (1–2) (2004) 81–90. [DOI] [PubMed] [Google Scholar]
  • [28].Kim M, Futai K, Jo J, Hayashi Y, Cho K, Sheng M, Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95, Neuron 56 (November (3)) (2007) 488–502. [DOI] [PubMed] [Google Scholar]
  • [30].Cerda O, Trimmer JS, Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels, Neurosci. Lett 486 (2) (2010) 60–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Du J, Haak LL, Phillips-Tansey E, Russell JT, McBain CJ, Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K+ channel subunit Kv2.1, J. Physiol 522 (Pt 1) (2000) 19–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Pascual JM, Shieh CC, Kirsch GE, Brown AM, Contribution of the NH2 terminus of Kv2.1 to channel activation, Am. J. Physiol 273 (6 Pt 1) (1997) C1849–C1858. [DOI] [PubMed] [Google Scholar]

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