Chronic Ca2+ influx through voltage-dependent Ca2+ channels enhance delayed rectifier K+ currents via activating Src family tyrosine kinase in rat hippocampal neurons

Yoon-Sil Yang; Sang-Chan Jeon; Dong-Kwan Kim; Su-Yong Eun; Sung-Cherl Jung

doi:10.4196/kjpp.2017.21.2.259

Journal List > Korean J Physiol Pharmacol > v.21(2) > 1026168

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Yang, Jeon, Kim, Eun, and Jung: Chronic Ca2+ influx through voltage-dependent Ca2+ channels enhance delayed rectifier K+ currents via activating Src family tyrosine kinase in rat hippocampal neurons

Original Article

The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology 2017; 21(2): 259-265.

Published online: 21 February 2017

DOI: https://doi.org/10.4196/kjpp.2017.21.2.259

Chronic Ca²⁺ influx through voltage-dependent Ca²⁺ channels enhance delayed rectifier K⁺ currents via activating Src family tyrosine kinase in rat hippocampal neurons

Yoon-Sil Yang^1,^¶, Sang-Chan Jeon¹, Dong-Kwan Kim³, Su-Yong Eun^1,², Sung-Cherl Jung^1,²

¹Department of Physiology, School of Medicine, Jeju National University, Jeju 63243, Korea.

²Institute of Medical Science, Jeju National University, Jeju 63243, Korea.

³Department of Physiology, College of Medicine, Konyang University, Daejeon 35365, Korea.

Correspondence: Sung-Cherl Jung. jungsc@jejunu.ac.kr

Current address:

^¶Present address: Department of Structure and Function of Neural Network, Korea Brain Research Institute, Daegu 41068, Korea

Received 21 December 2016 Revised 20 January 2017 Accepted 23 January 2017

(open-access, http://creativecommons.org/licenses/by-nc/4.0):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Excessive influx and the subsequent rapid cytosolic elevation of Ca²⁺ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca²⁺ level in normal as well as pathological conditions. Delayed rectifier K⁺ channels (I_DR channels) play a role to suppress membrane excitability by inducing K⁺ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca²⁺-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of I_DR channels to hyperexcitable conditions induced by high Ca²⁺ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca²⁺-treatment significantly increased the amplitude of I_DR without changes of gating kinetics. Nimodipine but not APV blocked Ca²⁺-induced I_DR enhancement, confirming that the change of I_DR might be targeted by Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I_DR enhancement was not affected by either Ca²⁺-induced Ca²⁺ release (CICR) or small conductance Ca²⁺-activated K⁺ channels (SK channels). Furthermore, PP2 but not H89 completely abolished I_DR enhancement under high Ca²⁺ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca²⁺-mediated I_DR enhancement. Thus, SFKs may be sensitive to excessive Ca²⁺ influx through VDCCs and enhance I_DR to activate a neuroprotective mechanism against Ca²⁺-mediated hyperexcitability in neurons.

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Keywords: A-type K⁺ channels, Delayed rectifier K⁺ channel, NMDA receptors, Src family tyrosine kinase, Voltage-dependent Ca²⁺ channel

INTRODUCTION

Dynamic changes of cytosolic Ca²⁺ level under physiological conditions are essential for cellular signalings of protein synthesis and signal transmission of neurons in central nervous system (CNS). However, due to its cytotoxic effects, excessive influx of Ca²⁺ and subsequent rapid elevation of [Ca²⁺]_i can induce irreversible damage of neurons or result in pathological dysfunctions such as epileptic seizure. Hence, mammalian neurons exhibit several protective mechanisms to homeostatically regulate membrane excitability and cytosolic Ca²⁺ level. Several types of voltage dependent ion channels are involved in these neuroprotective processes via regulating cation flow. In its original description based on neuronal excitability, intrinsic excitability can be defined as the ability to fire action potentials (APs), which are determined by voltage-dependent K⁺ and Na⁺ channels (K_V and Na_V channels) [1]. Therefore, cation channels especially sensitive to membrane potential are key factors to regulate excitability in most neurons even though they are inhibitory. In particular, several subtypes of Kv channels can determine resting membrane potential and effectively induce cation outflow to downregulate neuronal excitability in many conditions of membrane depolarization [2].

Among the many types of K⁺ channels expressed in CNS neurons, Kv2.1 is a major component of delayed rectifier K⁺ channel (I_DR channel), exhibiting sustained outward K⁺ currents [3 4 5]. This subtype plays a direct role in lowering membrane potential thus inhibiting AP initiation as well as limiting repetitive APs firing. Thus, the hyperpolarization of membrane potential and blockade of depolarization by Kv2.1 can prevent hyperexcitability that activates cytotoxic cascades and induces neuronal damage [6 7]. Previous studies showed that Kv2.1 channels are sensitive to changes in cytosolic Ca²⁺ level. For example, cytosolic Ca²⁺ increase and subsequent calcineurin activation affects the gating kinetics of I_DR channels via dephosphorylating Kv2.1 channels, since calcineurin-dependent dephosphorylation of Kv2.1 decreases the threshold for I_DR channel opening and disrupts channel clustering, resulting in changes of activation kinetics [1 8]. Kv2.1 channels have many serine, threonine, and tyrosine phosphorylation sites. These participate in the regulation of I_DR channel kinetics; for e.g., PKA-mediated phosphorylation in serine/threonine sites induces the changes of gating kinetics but not membrane expression of Kv2.1 channels [9 10]. Dynamic changes of gating kinetics by de- or phosphorylation of Kv2.1 channels seem to be minor but significant for regulating K⁺ outflow and membrane excitability under physiological conditions. However, other studies have recently demonstrated that under several pathological conditions such as epileptic seizures and ischemic damages, acute increment of cytosolic Ca²⁺ rapidly potentiates I_DR, and thus suppresses hyperexcitability [11 12 13]. These results suggest that rapid changes of cytosolic Ca²⁺ may stimulate more effective signaling pathways by which I_DR channels directly regulate membrane excitability. However, whether excessive Ca²⁺ influx rapidly and potentially enhances I_DR remains unclear.

In the present study, we confirmed that dissociated hippocampal neurons of rats showed significant increase of I_DR after high Ca²⁺ treatment, which could enhance synaptic activities and membrane excitability. The Ca²⁺-mediated I_DR upregulation was sensitive to activity of voltage-dependent Ca²⁺ channels (VDCCs) but not NMDA receptors (NMDARs), suggesting a neuroprotective signaling pathway that is not targeted by postsynaptic Ca²⁺ influx via NMDARs. In the additional experiment, we confirmed that the contribution of Src family tyrosine kinase (SFK) was necessary for upregulating I_DR under high Ca²⁺ condition, suggesting that the increased expression of I_DR channels via SFK activation is a protective mechanism against excitotoxicity and pathogenic processes in neurons.

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METHODS

Animal preparation and hippocampal primary culture

Hippocampal primary cultures were prepared from embryonic 20-day SD rats. After pregnant rats were deeply anesthetized, the embryos were removed and transferred to an ice-cold normal tyrode solution containing the following (in mM): 140 NaCl, 5.4 KCl, 2.3 MgCl₂, 10 HEPES, 5 glucose. The pH level was adjusted to 7.4 with NaOH. Isolated hippocampi from embryonic rat brains were transferred to ice-cold minimal essential medium (MEM) containing Earle's salts and glutamine with 10% fetal bovine serum, 0.45% glucose, 1 mM sodium pyruvate, 25 µM glutamate and antibiotics, and the hippocampi were triturated. The cells were counted and seeded on glass coverslips coated with poly-L-lysine (Sigma-Aldrich) at a density of 9×10⁴ cells/ml and transferred to an incubator gassed with 95% O₂ and 5% CO₂ at 37℃. After 7 hours, the whole plating medium was exchanged to Neurobasal medium (Sigma-Aldrich) containing B-27 (Invitrogen), and a half of the medium was replaced with new media once at DIV 5. Detail protocols for cell preparation and recording techniques were as previously reported [14 15]. All experiments and procedures with animals were approved by the Animal Care and Use Committee of Jeju National University.

Drugs treatment

To electrophysiologically observe the changes of I_DR in cultured hippocampal neurons, high Ca²⁺ condition was induced by doubling Ca²⁺ concentration of culture media (normal 1.8 mM) up to 3.6 mM (with CaCl₂, Sigma-Aldrich) for 24 hours. APV (100 mM, Sigma-Aldrich) and nimodipine (10 mM, Sigma-Aldrich) were used with high Ca²⁺ treatment to block NMDARs and VDCCs, respectively, in some experiments. H89 was added to block the activation of PKA and ryanodine receptors and IP₃ receptors of ER were blocked by Ryanodine and 2AP, respectively. Blockers of kinases and receptors were obtained from Sigma Aldrich. Caffeine or 4CMC (Sigma-Aldrich) were used to test the effects of CICR via ryanodine receptor activation. Details of drugs treatment are mentioned in results and legends of figures.

Electrophysiology

For patch-clamp recordings of dissociated hippocampal neurons, DIV 6~8 cultured cells were transferred to a recording chamber with a continuous flow of recording solution containing the following (in mM): 145 NaCl, 5 KCl, 2 CaCl₂, 1.3 MgCl₂, 10 HEPES, 10 glucose. pH level was adjusted to 7.4 with NaOH, and bubbled with 95% O₂ and 5% CO₂. TTX (0.5 µM, Tocrise) was added to the recording solution to block the Nav channels. The patch pipettes (4~6 MΩ) were filled with an internal solution containing the following (in mM): 20 KCl, 125 K⁺-gluconate, 4 NaCl, 10 HEPES, 0.5 EGTA, 4 ATP, 0.3 tris-GTP, and 10 phosphocreatin. pH and osmolarity were adjusted to 7.2 and 280~300 mOsm, respectively. In all experiments, neurons showing 7~11 pF whole cell capacitance were used. Whole cell recording parameters were monitored throughout each experiment and recordings where series resistance (6~20 MΩ) varied by more than 10% were rejected.

All recordings were performed at room temperature, low-pass filtered at 5 kHz, and digitized at 10 kHz by a Digidata 1322A convertor. Voltage-clamping whole cell patches, command pulse generation and data recording were performed using Axopatch 200B and pClamp 8 software. Thick-walled, filamented patch electrodes had tip resistance of 3~6 MOhm. For adjusting whole-cell parameters, membrane capacitance and series resistance were manually compensated in Axopatch 200B during applying command pulses. Series resistance varied between 8~30 MOhm, and recordings where series resistance varied by more than 10% were rejected. No electronic compensation for series resistance was employed during currents recording. Pulse commands to record sustained currents in voltage-clamping mode are described in detail in results and figures. Sustained K⁺ currents were digitally isolated using a prepulse protocol after subtracting leak currents. Peak currents were measured at +60 mV. All experimental data were additionally analyzed using IGOR pro software.

Statistical analysis

Further data and statistical analysis was performed using SPSS and Excel software and data were expressed as the mean value±standard error (SEM). The Student's t-test was used, and statistical difference between groups was indicated for p values of <0.05 or 0.01.

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RESULTS

In physiological regulation of neuronal excitability, the dominant role of small but sustained K⁺ outflow through I_DR channels is to determine and stabilize sub- and suprathreshold membrane potentials. However, signaling-mediated changes of its kinetics suggest a possibility that I_DR channels may also be targeted by hyperexcitable conditions. In the present study, we tested K⁺ outflow through I_DR channels in response to hyperexcitable condition induced by high Ca²⁺ overnight treatment (3.6 mM CaCl2, for 24 hours) in cultured hippocampal neurons. This experimental condition not only induces the downregulation of A-type K⁺ channels (I_A channels) but also enhances synaptic Ca²⁺ influx by remodeling NMDARs composition, resulting in the hyperexcitability of hippocampal neurons [14 16]. After treating high Ca²⁺ to culture media for 24 hours, neurons were transferred to a recording chamber and washed in normal ACSF solution for electrophysiological measurement. In results, the current density of I_DR channels was significantly increased by high Ca²⁺ treatment, as shown in Fig. 1 (Control=112.62±10.28 pA/pF, n=13; high Ca²⁺=171.05±18.30 pA/pF, n=12, p=0.02). This indicates that I_DR channels may be upregulated under hyperexcitable conditions.

Fig. 1

I_DR is enhanced under high Ca²⁺ condition in cultured hippocampal neurons, showing the dependence on VDCCs but not NMDARs.

(A) The experimental protocol of voltage clamping to record I_DR in hippocampal neurons and example traces of I_DR. High Ca²⁺ condition was induced by doubling Ca²⁺ concentration (CaCl₂, 3.6 mM) of culture media for 24 hours (High Ca²⁺) and then neurons were washed in normal ACSF during recording. Either APV (100 µM, APV+High Ca²⁺) or nimodipine (10 µM, Nimodipine + High Ca²⁺) was added to culture media for 24 hours under high Ca²⁺ condition. Scale bars 500 pA, 100 ms. (B) Individuals (circles) and averaged (square bars) densities of I_DR. Currents were adjusted with whole cell capacitance for measuring current densities of each group. Error bars represent SEM.

^*p<0.05.

Depolarization of membrane potential activates two major Ca²⁺ influx pathways: VDCC and NMDAR. High Ca²⁺-enhanced neuronal excitability is reportedly dependent on Ca²⁺ influx via NMDARs, hence it was necessary to confirm if the increase of I_DR was dependent on NMDARs [16]. APV (100 µM) or nimodipine (10 µM) with high Ca²⁺ treatment was applied to culture media for 24 hours before recording. APV-induced NMDARs block showed no effects on the high Ca²⁺-enhanced I_DR in this experiment; whereas, nimodipine, a VDCCs antagonist, significantly inhibited the effect of high Ca²⁺ treatment on I_DR (Fig. 1, APV=151.55±12.56 pA/pF, Nimodipine=96.03±9.34 pA/pF, p=0.48, 0.008 respectively, compared with high Ca²⁺). Thus, I_DR upregulation may be triggered by Ca²⁺ influx through VDCCs rather than NMDARs. Furthermore, because it is well known that Ca²⁺-induced Ca²⁺ release (CICR) is responsive to Ca²⁺ influx, we also tested if it might participate in I_DR upregulation resulted from Ca²⁺ influx via VDCCs. In Table 1, blocking of two major receptors of endoplasmic reticulum, ryanodine receptors (Ryanodine) and IP₃ receptors (2APB), showed no effects on enhanced I_DR under high Ca²⁺ condition. Additionally, caffeine or 4CMC, agonists of ryanodine receptors, showed no enhancement effects on I_DR, unlike high Ca²⁺ treatment. These results strongly indicated that Ca²⁺ influx through VDCCs may directly regulate I_DR channels regardless of CICR activation.

Table 1

The densities of I_DR recorded in hippocampal neurons under each condition

^*p<0.05, ^**p<0.01.

Although the current density of I_DR was increased, the gating kinetics of I_DR channels was not affected under high Ca²⁺ condition. In Fig. 2, the increase rate of I_DR positively reflected the increase rates of command pulse potentials in all experimental groups. At +80 mV injection, significant differences of I_DR amplitudes were observed between control and high Ca²⁺ treated neurons (Fig. 2A and B, Control=139.82±15.62 pA/pF; High Ca²⁺=226.79±17.34 pA/pF, p=0.02). The current density of I_DR under high Ca²⁺ condition was enhanced up to 60% in each injection range from −40 to 80 mV compared with the control group. However, in the analysis of conductance compensated by each membrane potential, activation properties of I_DR channels were consistent in all groups, indicating that the gating kinetics of I_DR channels may not be subjected to high Ca²⁺ condition (Fig. 2C and D, V_h of activation: Control=12.38±2.62 mV; High Ca²⁺=22.21±4.4 mV, p=0.1). This result suggests that the upregulation of I_DR channels under high Ca²⁺ condition may not be dependent on their dephosphorylation by calcineurin activated by Ca²⁺ signaling. Additionally, both antagonists, APV and nimodipine, showed no effects on the activation kinetics of I_DR channels (V_h of activation; APV group=10.11±1.91; Nimodipine group=14.92±1.63, p=0.55 and 0.45, respectively).

Fig. 2

The enhancement of I_DR under high Ca²⁺ condition does not involve the significant alteration of activation properties of I_DR channels.

The activation property was measured at −60 to 80 mV with 20 steps after prepulse injection (−20 mV, 200 ms duration). (A) The experimental protocol of voltage clamping to measure the activation kinetics of I_DR channels and example traces of I_DR. The amplitude of I_DR is increased by steps of voltage clamping. However, the constant rate of increment indicates no changes of I_DR activating kinetics. Scale bars 500 pA, 100 ms. (B) Averaged densities of I_DR at each clamping voltage (−40 to 80 mV). (C) and (D) Boltzmann fitted curves of gating kinetics of I_DR activation and averaged values of voltage half (V_h, dotted line in C). The significant change of activation curves was not observed. Error bars represent SEM.

Previously, Ledoux et al. [17] reported that the enhancement of intracellular Ca²⁺ concentration induced the elevation of K⁺ efflux through small conductance Ca²⁺ channels (SK channels). Because of possible change in intracellular Ca²⁺ concentration under the experimental condition, it was necessary to confirm whether the enhanced density of I_DR by high Ca²⁺ was contaminated by the flow of SK channels even though the two channels exhibit totally different gating mechanisms. In Fig. 3, the effect of 100 nM apamin with high Ca²⁺ was tested by adding to culture media for 24 hours to block SK channels. The addition of apamin did not influence the increased density of I_DR by high Ca²⁺ treatment, indicating that upregulating I_DR is not via the activation of SK channels (179.37±26.56 pA/pF, p=0.04 and 0.81, as compared with control and high Ca²⁺, respectively).

Fig. 3

SFKs are participated in I_DR enhancement under high Ca²⁺ condition.

(A) Example traces of I_DR in each group. To confirm the contribution of SK channels, apamin (100 nM, Ca²⁺+Apamin) was added to culture media. PP2 (1 µM) abolished the Ca²⁺-induced enhancement of I_DR, indicating the involvement of SFK in I_DR upregulation. (B) Individual (open circles) and averaged values (square bars) of I_DR densities. Error bars represent SEM. ^*p<0.05 compared with control and ^†p<0.05 compared with High Ca²⁺. (C) Averaged densities of I_DR at each clamping voltage (−40 to 80 mV). Error bars represent SEM.

Ca²⁺ signaling cascades activated by Ca²⁺ influx through synaptic NMDARs induce the phosphorylation of protein kinase A (PKA) resulting in the downregulation of I_A in hippocampal neurons [15 18]. Although it is still under argument, it is possible that PKA regulates the turnover rate of proteins from plasma membrane and currents through various channels. In this study, PKA was not involved in the cellular processing to enhance I_DR under high Ca²⁺ condition (Table 1, +H89). Rather, we confirmed that SFKs have a potentially important role in I_DR upregulation under high Ca²⁺ condition (Fig. 3). Adding SFK blocker, PP2 (1 µM), to culture media with high Ca²⁺ showed no enhancement of I_DR, with similar levels as in control neurons that were not treated with any drugs (Ca²⁺+PP2=99.01±10.52 pA/pF, p=0.43 and 0.02 compared with the control and high Ca²⁺, respectively). Furthermore, PP2 alone showed no effect on the density of I_DR, indicating that high Ca²⁺ condition possibly activate SFKs to prevent neuronal excitotoxicity or damage (123.46±20.23 pA/pF, p=0.63 compared with the control).

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DISCUSSION

Neurons in CNS exhibit dynamic regulatory signalings to protect from hyperexcitable conditions that result in neuronal damage. Herein, we reported upregulation of I_DR channels, as a neuroprotective regulator, which increased K⁺ outflow under hyperexcitable condition induced by high Ca²⁺ overnight treatment in cultured hippocampal neurons. The high Ca²⁺-induced enhancement of I_DR was dependent on Ca²⁺ influx through VDCCs but not NMDARs and was not affected by CICRs that synergistically increase intracellular Ca²⁺ level (Fig. 1 and Table 1). These results indicate that hippocampal neurons may homeostatically modulate I_DR channels in response to Ca²⁺ influx through VDCCs. Thus, neuronal excitability is consequently downregulated by the enhancement of K⁺ outflow via I_DR channels. Furthermore, I_DR enhancement was dependent on tyrosine kinases of Src family, indicating the possibility of an unique signaling cascade for the downregulation of excitability to prevent neuronal damages under hyperexcitable or pathogenic conditions.

High Ca²⁺ treatment for 24 hours used in this study (i.e. doubling Ca²⁺ concentration in culture media), is reported to mediate changes in the subunit composition of synaptic NMDARs, I_A density and Ca²⁺-calmodulin dependent kinase activity [14 16 19]. Increased Ca²⁺ influx through NMDARs and subsequent I_A downregulation critically increases neuronal excitability due to changes in membrane resistance, firing rates of APs and after-hyperpolarizing potentials resulting from direct effects of the cationic flow through channels or receptors [16 19]. These effects of high Ca²⁺ condition possibly result in irreversible damage to hippocampal neurons. However, hippocampal neurons showing Ca²⁺-induced downregulation of Kv4.2 channels had no effect on the resting membrane potential even though suprathreshold properties of membrane excitability were significantly changed [19]. This phenomenon is consistent with previous results in hippocampal neurons pretreated with high Ca²⁺, indicating that membrane potentials may also be targeted by a regulatory mechanism to prevent excitotoxicity [14]. In this study, the enhancement of I_DR strongly suggested a neuroprotective mechanism related with I_DR channels and Ca²⁺ influx through VDCCs (Fig. 1). I_DR channels contribute to the restoration of depolarized membrane potential to resting membrane potential in cardiac cells, determining refractory period as well as heart rate. In nervous system, these channels seem to contribute to the regulation of somatodendritic excitability. Kv2 family mediated with I_DR acts as a dominant regulatory factor for membrane excitability in hippocampal and cortical pyramidal neurons [20 21 22]. Experimental evidence indicates that the genetic manipulation of Kv2.1 directly affects intrinsic excitability, showing epileptiform activity, and that Kv2.1 knock-out mice exhibit defects in spatial learning [23]. These pathogenic outcomes are likely due to the hyperexcitability of neurons during development, as I_DR channels are targeted by cellular signaling pathways of various growth factors in young brain [24]. Therefore, enhanced I_DR under high Ca²⁺ condition observed in this study may be a rapid but effective neuroprotective modulation to prevent entry into pathogenic stages.

As a critical regulator of neuronal excitability, I_DR channels are dominantly targeted by Ca²⁺-mediated signaling in the manner of activity- and use-dependence. Even though I_DR channels are highly phosphorylated in neurons, the expression and gating kinetics of these channels are bidirectionally subjected to activity-dependent dephosphorylation, which is mediated by glutamatergic transmission [11 13 25]. The dephosphorylation of these channels can rapidly and homeostatically suppress neuronal excitability under pathological conditions via the enhancement of K⁺ outflow. Dephosphorylation of I_DR channels under anoxic stress is dependent on Ca²⁺ influx through NMDARs that are activated by excess glutamate release in synapses [25]. Thus, overexcitability of glutamatergic synapses in various pathological conditions possibly activates neuroprotective signalings to dephosphorylate and upregulate I_DR channels. However, we observed that enhanced I_DR under high Ca²⁺ condition was clearly dependent on Ca²⁺ influx through VDCCs but not NMDARs. This is evidenced by the effect of nimodipine to abolish the enhancement of I_DR (Fig. 1). Despite previous reports on the effects of VDCC blockers to inhibit I_DR, nimodipine, one of the dihydropyridines, showed no direct inhibition of I_DR in this study (data not shown) [26 27 28]. Because I_A downregulation is dependent on NMDARs under high Ca²⁺ condition, I_DR enhancement may be a possible mechanism of compensatory regulation in hippocampal neurons that are chronically exposed to high Ca²⁺ [14]. I_DR channels are predominantly regulated by calcineurin-dependent dephosphorylation, which is accompanied by changes of threshold as well as activation kinetics [1 8]. In this study, however, we confirmed that high Ca²⁺-induced upregulation of I_DR involved only the increase of I_DR peak but not the alteration of gating kinetics, suggesting the possibility of another signaling mechanism distinctive to calcineurin-dependent dephosphorylation. Moreover, the bidirectional and homeostatic upregulation of I_DR observed in neurons is due to the overexcitation of glutamatergic synapses, hence, the enhancement of I_DR peak without a kinetic change may be originated from Ca²⁺ influx via VDCCs rather than NMDARs [11 13 25].

The upregulation of I_DR peak without changes of gating kinetics suggests the involvement of specific modulatory signalings of kinases, which can be activated under high Ca²⁺ condition (Fig. 1 and 2). Previous reports focusing on regulatory mechanisms of KV2.1 channel indicate that these channels possess a large number of kinase binding sites such as serine, threonine, and tyrosine, and are usually affected by all except threonine [9 10]. In particular, tyrosine kinases, such as Src and Fyn, seem to play an important role in the regulation of KV2.1 channel expression. Although, tyrosine 124 phosphorylated by src regulates channels activities, tyrosine 810 phosphorylation influences the intracellular trafficking, regulating Kv2.1 channels expression [29 30 31]. Furthermore, growth differentiation factor 15 (GDF15)-induced upregulation of Kv2.1 channels expression was also abolished by the inhibition of Src-mediated phosphorylation of TGFβ receptors [24]. These previous reports indicate that SFKs are dominantly mediated with the cellular regulation of Kv2.1 channel expression, while the gating kinetics of I_DR channels is specifically targeted by serine kinases. Thus, the upregulation of I_DR by high Ca²⁺ treatment observed in this study may be originated from the modulatory function of tyrosine kinases. Fig. 2 clearly shows that PP2 blocked the enhancement of I_DR under high Ca²⁺ condition, indicative of SFKs' roles in I_DR channels for preventing hyperexcitability.

Because excessive Ca²⁺ influx is sufficient to cause hyper-excitability and irreversible damage to hippocampal neurons, resulting in pathological conditions, a number of subcellular signalings in neurons target the expression and gating kinetics of Kv channels which actively regulate membrane excitability. The current study confirmed that a neuroprotective regulatory mechanism involving I_DR channels in hippocampal neurons possibly plays a role to suppress neuronal excitability under various pathogenic conditions. Although further study is needed to clarify the relationship(s) between Ca²⁺ influx via VDCCs and SFK signaling in I_DR upregulation, VDCC-mediated upregulation of I_DR suggests another signaling pathway to actively regulate membrane excitability of hippocampal neurons.

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ACKNOWLEDGEMENTS

This work was supported by the Academic Research Foundation of Jeju National University Institute of medical science in 2014.

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Notes

Author contributions: Y.S.Y. and S.C.J. performed electrophysiological experiments. Y.S.Y. and D.K.K. performed statistical analysis. S.Y.E. and Y.S.Y. drafted the manuscript. S.C.J. designed, supervised and coordinated the study and wrote the manuscript.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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