Neuroscience Letters 583 (2014) 142–147
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
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 a b
Neuroscience Center, Louisiana State University Health Science Center, New Orleans, LA, United States Department of Structural and Cellular Biology, School of Medicine, Tulane University, New Orleans, LA, United States
h i g h l i g h t s • 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 .
a r t i c l e
i n f o
Article history: Received 31 March 2014 Received in revised form 25 August 2014 Accepted 29 August 2014 Available online 8 September 2014 Keywords: Kv2.1 NMDA receptor Synaptic activity Dephosphorylation PP1
a b s t r a c t 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. © 2014 Published by Elsevier Ireland Ltd.
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
∗ Corresponding author. Current address: The Scripps Research Institute, 10550 North Torrey Pines Road, DNC-118, La Jolla, CA 92037, United States. Tel.: +1 858 784 1000. E-mail address: [email protected] (B. Siddoway). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.08.051 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
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
B. Siddoway et al. / Neuroscience Letters 583 (2014) 142–147
143
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 E18 SD rat embryos (cortex/hippocampus). Cells were plated at 0.5 × 106 per well in six well plates (∼50 K/cm2 for biochemistry, 100 K/cm2 for electrophysiology) on poly-l-lysine (50 g/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
Fig. 1. Protein phosphatase-1 dephosphorylates Kv2.1 in response to synaptic activity. (A) Neurons were pretreated with 1 М FK506 + 20 М cyclosporinA (CSA) or 1 М FK506 + 20 М CSA + 500 nМ okadaic acid for 20 min and 100 М NMDA was added for 10 min. (B) PP1 was recombinantly expressed in neurons, 100 М 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 М bicuculline and 500 М 4-AP was added for 2 h. (D) To induce dephosphorylation of Kv2.1, 40 М bicuculline or 40 М bicuculline and 500 М 4-AP was added to neurons for 2 h, neurons were pretreated for 20 min with 2 М 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 М bicuculline and 500 М 4-AP was added to neurons at the times indicated.
144
B. Siddoway et al. / Neuroscience Letters 583 (2014) 142–147
(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. 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 M 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
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
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 a b
Neuroscience Center, Louisiana State University Health Science Center, New Orleans, LA, United States Department of Structural and Cellular Biology, School of Medicine, Tulane University, New Orleans, LA, United States
h i g h l i g h t s • 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 .
a r t i c l e
i n f o
Article history: Received 31 March 2014 Received in revised form 25 August 2014 Accepted 29 August 2014 Available online 8 September 2014 Keywords: Kv2.1 NMDA receptor Synaptic activity Dephosphorylation PP1
a b s t r a c t 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. © 2014 Published by Elsevier Ireland Ltd.
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
∗ Corresponding author. Current address: The Scripps Research Institute, 10550 North Torrey Pines Road, DNC-118, La Jolla, CA 92037, United States. Tel.: +1 858 784 1000. E-mail address: [email protected] (B. Siddoway). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.08.051 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
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
B. Siddoway et al. / Neuroscience Letters 583 (2014) 142–147
143
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 E18 SD rat embryos (cortex/hippocampus). Cells were plated at 0.5 × 106 per well in six well plates (∼50 K/cm2 for biochemistry, 100 K/cm2 for electrophysiology) on poly-l-lysine (50 g/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
Fig. 1. Protein phosphatase-1 dephosphorylates Kv2.1 in response to synaptic activity. (A) Neurons were pretreated with 1 М FK506 + 20 М cyclosporinA (CSA) or 1 М FK506 + 20 М CSA + 500 nМ okadaic acid for 20 min and 100 М NMDA was added for 10 min. (B) PP1 was recombinantly expressed in neurons, 100 М 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 М bicuculline and 500 М 4-AP was added for 2 h. (D) To induce dephosphorylation of Kv2.1, 40 М bicuculline or 40 М bicuculline and 500 М 4-AP was added to neurons for 2 h, neurons were pretreated for 20 min with 2 М 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 М bicuculline and 500 М 4-AP was added to neurons at the times indicated.
144
B. Siddoway et al. / Neuroscience Letters 583 (2014) 142–147
(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. 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 M 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
