European Journal of Pharmacology 855 (2019) 175–182

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European Journal of Pharmacology

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Full length article

Effects of cenobamate (YKP3089), a newly developed anti-epileptic drug, on T

voltage-gated sodium channels in rat hippocampal CA3 neurons
Michiko Nakamuraa,b, Jin-Hwa Choa, Hyewon Shinc, Il-Sung Janga,b,∗
a
Department of Pharmacology, School of Dentistry, Kyungpook National University, 2177 Dalgubeol-daero, Jung-gu, Daegu, 700-412, Republic of Korea
b
Brain Science & Engineering Institute, Kyungpook National University, 2177 Dalgubeol-daero, Jung-gu, Daegu, 700-412, Republic of Korea
c
Department of Pharmacology, SK Biopharmaceuticals, Co., Ltd., 221 Pangyoyeok-ro, Seongnam, Gyeonggi, 305-712, Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: New, more effective pharmacologic treatments for epilepsy are needed, as a substantial portion of patients
Epilepsy (> 30%) are refractory to currently available anti-epileptic drugs. Cenobamate (YKP3089) is an investigational
Anti-epileptic drugs anti-epileptic drug in clinical development. Two completed adequate and well-controlled studies demonstrated a
Voltage-gated Na+ channels significant reduction in focal seizures with cenobamate in patients with epilepsy. In this study, we characterized
Hippocampal neurons
the effects of cenobamate on voltage-gated Na+ channels in acutely isolated rat hippocampal CA3 neurons using
Patch clamp
a whole-cell patch-clamp technique. While cenobamate had little effect on the peak component of transient Na+
current (INaT) induced by brief depolarizing step pulses, it potently inhibited the non-inactivating persistent
component of INa (INaP). In addition, cenobamate potently inhibited the current by slow voltage-ramp stimuli.
Cenobamate significantly shifted the steady-state fast inactivation relationship toward a hyperpolarizing range,
indicating that cenobamate binds to voltage-gated Na+ channels at the inactivated state with a higher affinity.
Cenobamate also accelerated the development of inactivation and retarded recovery from inactivation of vol-
tage-gated Na+ channels. In current clamp experiments, cenobamate hyperpolarized membrane potentials in a
concentration-dependent manner, and these effects were mediated by inhibiting the INaP. Cenobamate also in-
creased the threshold for generation of action potentials, and decreased the number of action potentials elicited
by depolarizing current injection. Given that the INaP plays a pivotal role in the repetitive and/or burst gen-
eration of action potentials, the cenobamate-mediated preferential blockade of INaP might contribute to anti-
epileptic activity.

1. Introduction existence of slowly inactivating or non-inactivating Na+ currents

Epilepsy is a common neurological disorder characterized by re-
current and unprovoked seizures (Brodie et al., 2012; Chen et al., 2018;
Kwan and Brodie, 2000; Tian et al., 2018). Excessive excitability in
neural tissues is believed to contribute to epilepsy (Das et al., 2012).
Anti-epileptic drugs (AEDs) acting on voltage-gated Na+ channels have
long been utilized for the pharmacologic treatment of epilepsy because
voltage-gated Na+ channels play a pivotal role in the generation and
conduction of action potentials (Catterall, 2014). However, despite the
use of many currently available AEDs, seizures remain uncontrolled in
over 30% of patients with epilepsy (Brodie et al., 2012; Chen et al.,
2018; Kwan and Brodie, 2000; Tian et al., 2018).
Transient Na+ currents (INaT) are generally quickly activated and
then inactivated upon membrane depolarization. However, the
mediated by voltage-gated Na+ channels, called persistent Na+ cur-
rents (INaP), has been identified in central as well as peripheral neurons
(Crill, 1996; Wu et al., 2005; Xie et al., 2011). In addition to its role in
the excitability and firing pattern in neurons of many brain regions
(Bennett et al., 2000; Jackson et al., 2004; Taddese and Bean, 2002; Yue
et al., 2005), the INaP plays a crucial role in several pathological con-
ditions, such as epilepsy and pain (Hains and Waxman, 2007; Lossin
et al., 2002; Stafstrom, 2007). In fact, several point mutations in vol-
tage-gated Na+ channels that exhibit increased INaP have been identi-
fied in patients with epilepsy (Lossin et al., 2002; Stafstrom, 2007).
Moreover, in subicular burst firing neurons resected from patients with
temporal lobe epilepsy, a large increase of INaP has been recorded, with
an amplitude up to half of the total sodium current (Vreugdenhil et al.,
2004). Several AEDs targeting voltage-gated Na+ channels, such as

∗
Corresponding author. Professor Department of Pharmacology, School of Dentistry Kyungpook National University, 2177 Dalgubeol-daero, Jung-gu, Daegu, 700-
412, Republic of Korea.
E-mail addresses: michiko21a@hotmail.com (M. Nakamura), cjinhwa@knu.ac.kr (J.-H. Cho), hyewon.shin@sk.com (H. Shin), jis7619@knu.ac.kr (I.-S. Jang).

https://doi.org/10.1016/j.ejphar.2019.05.007
Received 6 December 2018; Received in revised form 30 April 2019; Accepted 3 May 2019
Available online 04 May 2019
0014-2999/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
M. Nakamura, et al.
phenytoin, valproic acid, and lamotrigine, are known to inhibit this
increased INaP to some extent (Spadoni et al., 2002; Stafstrom, 2007). A
more recent study has also shown the anti-epileptic activity of two
compounds that preferentially block INaP in animal models (Anderson
et al., 2014), indicating that the INaP may be a promising target for the
development of new AEDs.
Cenobamate (YKP3089) is an investigational AED that has shown a
broad-spectrum of anticonvulsant activity in rodent epilepsy models
(Bialer et al., 2013). In mice and rats, cenobamate displayed an antic-
onvulsant activity in the maximal electroshock test and prevented sei-
zures induced by chemical convulsants such as pentylenetetrazol and
picrotoxin. In addition, cenobamate was reported to be effective in two
models of focal seizure, the hippocampal kindled rat and the mouse
6 Hz psychomotor seizure models. Two completed adequate and well-
controlled clinical studies demonstrated a significant reduction in focal
seizures with cenobamate in patients with epilepsy (clinicaltrials.gov
NCT01397968 and NCT01866111), and a long-term open-label phase 3
safety clinical trial is currently ongoing (clinicaltrials.gov
NCT02535091). In this study, we characterized the mode of action of
cenobamate in acutely isolated rat hippocampal CA3 pyramidal neu-
rons using a whole-cell patch-clamp technique.
2. Materials and methods
2.1. Preparation
All experiments complied with the guiding principles for the care
and use of animals approved by the Kyungpook National University and
Council of the Physiological Society of Korea, and every effort was
made to minimize both the number of animals used and their suffering.
Sprague Dawley rats (12–17 day-old, either sex) were decapitated
under ketamine anesthesia (100 mg/kg, i.p.). The brain was dissected
and transversely sliced at a thickness of 400 μm using a microslicer
(VT1000S; Leica, Nussloch, Germany). Slices containing the hippo-
campus were kept in an incubation medium (in mM; 124 NaCl, 3 KCl,
1.5 KH2PO4, 24 NaHCO3, 2 CaCl2, 1.3 MgSO4 and 10 glucose) saturated
with 95% O2 and 5% CO2 at room temperature (22-24 °C) for at least
1 h before the mechanical dissociation. For dissociation, slices were
transferred into a 35 mm culture dish (Primaria 3801; Becton
Dickinson, Rutherford, NJ, USA), which contained a standard external
solution (in mM; 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10
HEPES, adjusted to a pH of 7.4 with Tris-base), and the hippocampal
CA3 region was identified under a binocular microscope (SMZ-1; Nikon,
Tokyo, Japan). Details of the mechanical dissociation have been de-
scribed previously (Akaike and Moorhouse, 2003; Rhee et al., 1999).
Briefly, mechanical dissociation was accomplished using a custom-built
vibration device and a fire-polished glass pipette oscillating at about
50–60 Hz (0.3–0.5 mm) on the surface of the CA3 pyramidal layer.
Slices were removed and mechanically dissociated neurons allowed to
settle and to adhere to the bottom of the dish for 15 min. These dis-
sociated neurons lost the most distal processes, but a short portion
(∼100 μm in a length) of their thick proximal dendrites was retained,
as previously shown (Jang et al., 2006) (Fig. 1A).
2.2. Electrophysiology
All electrical measurements were performed using conventional
whole-cell patch recordings and a patch-clamp amplifier (Axopatch
200B; Molecular Devices, Union City, CA, USA) with a K+-free external
solution (in mM; 130 NaCl, 20 tetraethylammonium-Cl, 3 CsCl, 2 CaCl2,
1 MgCl2, 10 glucose, and 10 HEPES, adjusted to a pH of 7.4 with Tris-
base). In a subset of experiments using a low Na+ external solution,
100 mM NaCl was replaced with equimolar N-methyl-D-glucamine-Cl.
Neurons were voltage-clamped at a holding potential (VH) of −80 mV,
except where indicated. Patch pipettes were made from borosilicate
capillary glass (G-1.5; Narishige, Tokyo, Japan) using a pipette puller
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European Journal of Pharmacology 855 (2019) 175–182
(P-97; Sutter Instrument Co., Novato, CA, USA). The resistance of the
recording pipettes filled with the internal solution (in mM; 140 CsF, 10
CsCl, 2 EGTA, 2 ATP-Na2, and 10 HEPES, with the pH adjusted to 7.2
with Tris-base) was 0.8–1.5 MΩ. In current-clamp experiments, 140 CsF
and 10 CsCl were replaced with equimolar 140 KF and 10 KCl, re-
spectively. Membrane potentials were corrected for the liquid junction
potential. Neurons were viewed under phase contrast on an inverted
microscope (TE-2000; Nikon, Tokyo, Japan). Membrane currents were
filtered at 2–5 kHz, digitized at 10–20 kHz, and stored on a computer
equipped with pCLAMP 10.3 (Molecular Devices). Capacitative and
leakage currents were subtracted by the P/4 protocol using pCLAMP
program. During recordings, 10 mV hyperpolarizing step pulses (30 ms
in duration) were periodically applied to monitor the access resistance,
and recordings were discontinued if access resistance changed by more
than 10%. All experiments were performed at room temperature
(22°C–25 °C). To record voltage-gated Na+ currents (INa), the K+-free
external solution routinely contained 100 μM Cd2+ to block voltage-
gated Ca2+ channels. Depolarizing step pulses to evoke the INa were
applied with an interval of 10 s (except where indicated), which was
sufficiently long enough to recover from the inactivation of voltage-
gated Na+ channels. To record membrane currents of ligand-gated ion
channels, CsF within the pipette solution was replaced with equimolar
Cs-methanesulfonate.
2.3. Data analysis
The amplitude of INa was measured by subtracting the baseline from
the peak amplitude of INa by using pCLAMP 10.3 (Molecular Devices).
The continuous curves for the concentration-response relationship were
fitted using a least-squares fit method to the following equation, I = 1 –
[Cn/(Cn + IC50n)], where I is the relative amplitude of INa changed by
cenobamate, C is the cenobamate concentration, IC50 is the cenobamate
concentration for the half-maximal response, and n is the Hill coefficient.
The amplitude of INa was transformed into conductance (G) using the
following equation; G = I/(V – ENa), where ENa is the Na+ equilibrium
potential calculated by the Nernst equation. The voltage-activation and
voltage-inactivation relationships of Na+ channels were fitted to the
Boltzmann equations, respectively; G/Gmax = 1/{1 + exp[(V50,act – V)/
k]} and I/Imax = 1–1/{1 + exp[(V50,inact – V)/k]}, where Gmax and Imax
are the maximum conductance and current amplitude, respectively,
V50,act and V50,inact are half-maximal potentials for activation and fast
inactivation, respectively, and k is the slope factor. The fast (τfast) and
slow (τslow) time constants of the decay of single INa and the kinetic data
for the recovery from inactivation were fitted to the following equation; I
(t) = A0 + Afast x [1 – exp(–t/τfast)] + Aslow x [1 – exp(–t/τslow)], and the
kinetic data for the development of inactivation were fitted to the fol-
lowing equation; I(t) = A0 + Afast x [exp(–t/τfast)] + Aslow x [exp(–t/
τslow)], where I(t) is the amplitude of INa at time t, and Afast and Aslow are
the amplitude fraction of τfast, and τslow, respectively. The weighted time
constant (τWD or τweighted) was calculated by using the following equa-
tion: τWD or τweighted = [(τfast x Afast) + (τslow x Aslow)]/(Afast + Aslow).
The binding affinity of cenobamate for resting or inactivated Na+
channels was fitted to the following equation (Bean, 1984): exp
(ΔV50,inact/k) = [1 + (C/KI)]/[1 + (C/KR)], where ΔV50,inact, C, KI, and
KR represent the voltage shift, cenobamate concentration, dissociation
constants for inactivated and resting states, respectively. Numerical va-
lues are provided as the mean ± S.E.M. using values normalized to the
control. Significant differences in the mean amplitude were tested using
Student's paired two-tailed t-test, except where indicated, with absolute
values rather than normalized ones. Values of P < 0.05 were considered
significantly different.
2.4. Drugs
The drugs and chemicals used in the present study were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Cenobamate (Batch PD187-
M. Nakamura, et al.
14-002N) was provided by SK Biopharmaceuticals, Co., Ltd (Gyeonggi,
Republic of Korea). Cenobamate (YKP3089) was dissolved in dimethyl
sulfoxide to give a stock solution of 100–300 mM, and the final con-
centration of dimethyl sulfoxide applied to the external solution
was < 0.1% v/v. All solutions containing drugs were applied using the
“Y-tube system” for rapid solution exchange (Murase et al., 1989).
3. Results
3.1. Effects of cenobamate on voltage-gated Na+ channels in acutely
isolated hippocampal CA3 neurons
INa were induced by depolarizing step pulses (−80 mV to −20 mV,
every 5 s) in acutely isolated hippocampal CA3 neurons. The INaT was
quickly decayed with a time constant of 0.40 ± 0.01 ms (n = 7, Fig. 1B
and C), but the INa was not completely returned to baseline even after
200 ms of sustained depolarizing stimuli (Fig. 1B). The transient com-
ponent of INa was 10.6 ± 1.6 nA (n = 22), and the persistent compo-
nent of INa was 135.1 ± 21.8 pA (n = 22) (Fig. 1B). This persistent
component during sustained depolarizing stimuli was similar to the
non-desensitizing or INaP shown in previous studies (Kohling, 2002;
Urbani and Belluzzi, 2000). We next examined the effects of cen-
obamate on INa in acutely isolated rat hippocampal CA3 neurons.
Cenobamate (100 μM) hardly affected the INaT amplitude, as shown by a
slight decrease to 94.6 ± 2.5% of vehicle control (n = 7, P < 0.01,
Fig. 1B and D). In contrast, cenobamate (100 μM) potently decreased
the INaP to 25.6 ± 4.4% of control (n = 7, P < 0.01, Fig. 1B and D).
The cenobamate-mediated inhibition of the transient and persistent
components was concentration-dependent (IC50 > 500 μM for the INaT,
and IC50 = 53.1 ± 4.2 μM for the INaP, n = 7, respectively, Fig. 1D).
Cenobamate (100 μM) had no effect on the time to peak or the weighted
decay time constant (τWD) of transient INa (time to peak; 0.40 ± 0.01
for control and 0.41 ± 0.02 for cenobamate, n = 7, P = 0.88, τWD;
1.1 ± 0.04 for control and 1.0 ± 0.03 for cenobamate, n = 7,
P = 0.36, Fig. 1C). Cenobamate produced a more potent and pre-
ferential inhibition of INaP current compared with carbamazepine and
lamotrigine (Supplementary Fig. S1).
3.2. Effect of cenobamate on Na+ currents by slow voltage-ramps
The membrane currents induced by the slow voltage-ramp com-
mand (−80 mV to +10 mV, 6 s, in every 10 s) were recorded from
hippocampal CA3 neurons. These slowly inactivating currents were
completely blocked either by 10 μM riluzole (Fig. 2Aa), which is known
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European Journal of Pharmacology 855 (2019) 175–182
Fig. 1. Effects of cenobamate on voltage-
gated Na+ currents in acutely isolated hip-
pocampal CA3 neurons. A: A typical phase
contrast image of an acutely isolated CA3
pyramidal neuron. B: Typical traces of INaT
and INaP in the absence and presence of
100 μM cenobamate. C: Onset time (a) and
decay time constant (b) of INaT in the ab-
sence (open column) and presence (closed
column) of 100 μM cenobamate. Each
column represents the mean ± S.E.M. from
7 experiments (7 animals). n.s., not sig-
nificant. D: Concentration-response re-
lationships of cenobamate against the peak
(open circles) and steady state components
(closed circles) of INaT. Each point re-
presents the mean ± S.E.M. from 7 ex-
periments (7 animals).
to inhibit persistent currents but not transient Na+ currents at central
neurons (Urbani and Belluzzi, 2000), or 300 nM tetrodotoxin, a specific
voltage-gated Na+ channel blocker (Fig. 2Ab), suggesting that the slow
voltage-ramp-induced membrane currents share properties with INaP
mediated by voltage-gated Na+ channels as shown in previous studies
(Bellingham, 2011; Park et al., 2013; Parri and Crunelli, 1998). Next,
the effects of cenobamate on the currents by slow voltage-ramps in
hippocampal CA3 neurons were examined. Cenobamate decreased the
amplitude of the currents in a concentration-dependent manner
(IC50 = 53.1 ± 4.8 μM, n = 6). At 100 μM cenobamate, the current
amplitude decreased to 31.5 ± 1.4% of control (n = 6, P < 0.01,
Fig. 2B and D). The conductance of currents recorded at each voltage of
ramp command was calculated and normalized to the maximal con-
ductance in the control condition, and the data were fitted to the
Boltzmann function (Fig. 2C). Cenobamate (100 μM) did not shift V50,act
(−47.8 ± 3.5 for control and −51.1 ± 4.0 mV for cenobamate,
n = 6, P = 0.45, Fig. 2C). In addition, cenobamate did not change the
slope factor of voltage dependence (Fig. 2Cb).
3.3. Effects of cenobamate on the voltage dependence of Na+ channels
The effect of cenobamate on the voltage-activation relationship of
Na+ channels was examined. In these experiments, extracellular Na+
concentration ([Na+]o) was decreased to 30 mM to improve the quality
of the voltage-clamp. In these conditions, the INa was induced by 50 ms
depolarizing test pulses from −80 to +30 mV in 10 mV increments
before and during application of cenobamate (Fig. 3A). However,
cenobamate at a concentration of 100 μM concentration had little effect
on the current-voltage relationship (Fig. 3B). When the conductance at
each voltage was normalized to the maximal conductance in the control
condition, and the pooled data were fitted to the Boltzmann function,
cenobamate concentrations at ≤100 μM did not change the half-max-
imal voltage for activation (V50,act) (−38.2 ± 4.1 mV and
−37.9 ± 4.3 mV in the absence and presence of 100 μM cenobamate,
0.3 ± 0.2 mV shift, n = 8, P = 0.41, Fig. 3C and D). In addition, cen-
obamate did not change the slope factor of voltage dependence (data
not shown).
Next, the effect of cenobamate on the voltage dependence for the
steady-state fast inactivation of Na+ channels was examined. The INa
was induced by 50 ms depolarization test pulses to −20 mV after
500 ms prepulses from −120 to −30 mV in 10 mV increments
(Fig. 3E). The peak amplitudes of INa recorded at each prepulse voltages
were normalized to the maximal currents in the control condition, and
the data were fitted to the Boltzmann function (Fig. 3E and F).
M. Nakamura, et al. European Journal of Pharmacology 855 (2019) 175–182

Fig. 2. Effects of cenobamate on persistent Na+

currents in hippocampal CA3 neurons. A: Typical
traces of currents in the absence and presence of
10 μM riluzole (a) and 300 nM tetrodotoxin (TTX, b)
induced by slow voltage-ramp commands (15 mV/s).
The slow voltage-ramp current was obtained by
subtraction of raw traces (a–b). B: Typical traces of
slow voltage-ramp current in the absence and pre-
sence of 100 μM cenobamate. Ca: Conductance-vol-
tage relationships of voltage-gated Na+ channels in
the absence (open circles) and presence (closed cir-
cles) of 100 μM cenobamate. Each point represents
the mean ± S.E.M. from 6 experiments. The con-
ductance was calculated by using the following
equation: G = I/(V – Vrev), where I is the amplitude
of the INaP and Vrev is the reversal potential of Na+.
Cb: Effects of cenobamate (100 μM) on the midpoint
voltage for activation (V50,act, left) and slope factor.
Each column represents the mean ± S.E.M. from 6
experiments (6 animals). n.s., not significant. D:
Concentration-response relationships of cenobamate
against the INaP. Each point represents the
mean ± S.E.M. from 6 experiments (6 animals).

Cenobamate shifted the midpoint voltage for inactivation (V50,inact)
toward a hyperpolarizing range in a concentration-dependent manner;
100 μM cenobamate shifted the V50,inact from −59.1 ± 3.1 to
−65.0 ± 2.6 mV (−5.9 ± 2.6 mV shift, n = 8, P < 0.01) (Fig. 3F
and G). However, cenobamate did not change the slope factor of vol-
tage dependence for steady-state fast inactivation (5.5 ± 0.2 for
control and 6.1 ± 0.4 for cenobamate, n = 8, P = 0.19). It should be
noted that cenobamate did not reduce the INa at hyperpolarized mem-
brane potentials (−120 to −100 mV) (Fig. 3F). When the extent of
cenobamate-induced hyperpolarizing shift was plotted and fitted (see
Methods), the corresponding KI and KR values of cenobamate were
48.2 ± 6.8 μM and 797.7 ± 155.4 μM, respectively (Fig. 3H).

Fig. 3. Effects of cenobamate on the current-voltage relationship of voltage-gated Na+ channels in hippocampal CA3 neurons. Aa: A schematic illustration of voltage
step pulses. The INa was induced by 50 ms depolarization pulses from −80 to +30 mV in 10 mV increments at a VH of
−80 mV. Ab: Typical traces of the INa induced by voltage step pulses in the absence (left) and presence (right) of 100 μM cenobamate. Each point represents the
mean ± S.E.M. from 8 experiments (8 animals). B: Current-voltage relationships of voltage-gated Na+ channels in the absence (open circles) and presence (closed
circles) of 100 μM cenobamate. Each point represents mean ± S.E.M. from 8 experiments (8 animals). C: Conductance-voltage relationships of voltage-gated Na+
channels in the absence (open circles) and presence (closed circles) of 100 μM cenobamate. Each point represents the mean ± S.E.M. from 8 experiments. The
conductance was calculated by using the following equation: G = I/(V – Vrev), where I is the peak amplitude of the INa and Vrev is the reversal potential of Na+. D:
Cenobamate-induced changes in the midpoint voltage for activation (V50,act) of voltage-gated Na+ channels. Each column represents the mean ± S.E.M. from 8
experiments (8 animals). *P < 0.05; n.s., not significant. Note that cenobamate at ≤100 μM concentrations did not change the midpoint voltage for activation. Ea: A
schematic illustration of voltage step pulses. The INa was induced by 50 ms depolarization pulses to −20 mV after 500 ms prepulses from −120 to −30 mV in 10 mV
increments. Eb: Typical traces of the INa induced by voltage step pulses in the absence (left) and presence (right) of 100 μM cenobamate. Each point represents the
mean ± S.E.M. from 8 experiments (8 animals). F: Current-voltage relationships of voltage-gated Na+ channels in the absence (open circles) and presence (closed
circles) of 100 μM cenobamate. Each point represents the mean ± S.E.M. from 8 experiments (8 animals). G: Cenobamate-induced changes in the midpoint voltage
for inactivation (V50,inact) of voltage-gated Na+ channels. Each column represents the mean ± S.E.M. from 8 experiments (8 animals). **P < 0.01; n.s., not
significant. H: The exp(ΔV50,inact/k) values were plotted against the cenobamate concentration. Each point represents the mean ± S.E.M. from 6 to 8 experiments (8
animals). The KR and KI values were 797.7 μM and 48.2 μM, respectively.

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M. Nakamura, et al.
τslow, Afast, Aslow, and τweighted) for the development of inactivation of voltage-gated Na+ channels. Each column represents the mean ± S.E.M. from 9 experiments
(9 animals). *P < 0.05, **P < 0.01.
3.4. Effect of cenobamate on inactivation kinetics of voltage-gated Na+
channels
The effects of cenobamate on the development of inactivation of
voltage-gated Na+ channels were examined by use of the two-pulse
protocol, where the first conditioning pulse (P1) varying from 2 to
8000 ms in duration was followed by a second test pulse (P2) with an
interpulse interval of 20 ms (Fig. 4A and B). The fraction of voltage-
gated Na+ channels available in the second test pulse was determined
as the P2/P1 ratio. Then, the P2/P1 ratio was plotted against the dura-
tion of first conditioning pulse. The extent of the development of in-
activation was well-fitted to the double exponential function with the
fast (τfast) and slow time constants (τslow) (Fig. 4C). Cenobamate
(100 μM) significantly decreased the τfast and τslow to 47.4 ± 3.2% and
66.6 ± 7.8% of control, respectively (τfast; 445.2 ± 76.1 ms for con-
trol and 211.2 ± 12.8 ms for cenobamate, τslow; 6097.8 ± 631.3 ms
for control and 4059.2 ± 454.5 ms for cenobamate, n = 9, P < 0.05,
respectively, Fig. 4D). Cenobamate (100 μM) also decreased the
weighted time constant (τweighted) to 39.1 ± 7.9% of control
(5584.7 ± 551.3 ms for control and 2126.4 ± 257.0 ms for cen-
obamate, n = 9, P < 0.01, Fig. 4D), and induced statistically sig-
nificant decreases in Afast and Aslow (Afast; 0.67 ± 0.01 for control and
0.61 ± 0.01 for cenobamate, n = 9, P < 0.05, Aslow; 0.34 ± 0.02 for
control and 0.23 ± 0.02 for cenobamate, n = 9, P < 0.01, respec-
tively, Fig. 4D).
The effects of cenobamate on the recovery from inactivation of
voltage-gated Na+ channels were examined by use of the two-pulse
protocol, where Na+ channels were fully inactivated by the first pulse
(P1, 500 ms duration) and allowed to recover during interpulse inter-
vals varying from 1 to 5000 ms before applying the second test pulse
(P2, 50 ms duration) (Fig. 5A and B). The fraction of voltage-gated Na+
channels available in the second test pulse was determined as the P2/P1
ratio. And then, the P2/P1 ratio was plotted against the duration of
interpulse intervals. The extent of the recovery from inactivation was
well-fitted to the double exponential function (Fig. 5C). Cenobamate
(100 μM) significantly increased the τfast and τslow to 248.9 ± 27.7%
and 523.7 ± 66.2% of the control, respectively (τfast, 4.7 ± 0.3 ms for
control and 11.7 ± 1.1 ms for cenobamate; τslow, 25.7 ± 3.1 ms for
control and 134.6 ± 11.8 ms for cenobamate, n = 5, P < 0.01, re-
spectively, Fig. 5D). Cenobamate (100 μM) also increased the τweighted
to 520.2 ± 34.7% of control (12.0 ± 1.0 ms for control and
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European Journal of Pharmacology 855 (2019) 175–182
Fig. 4. Effect of cenobamate on the onset of
inactivation of voltage-gated Na+ channels
in hippocampal CA3 neurons. A: A sche-
matic illustration of voltage step pulses. The
INa was induced by the two-pulse protocol,
where the first conditioning pulses (P1; −20
mV depolarization, 2–8000 ms duration)
were followed by the second test pulses (P2;
−20 mV depolarization, 50 ms duration).
The second INa was recovered with an in-
terpulse interval of 20 ms at a potential of
−80 mV. B: Typical first (P1) and second
(P2) traces of INa induced by the two-pulse
protocol in the absence (left) and presence
(right) of 100 μM cenobamate. C: Kinetics
for the development of inactivation of vol-
tage-gated Na+ channels in the absence
(open circles) and presence (closed circles)
of 100 μM cenobamate. Each point re-
presents the mean ± S.E.M. from 9 ex-
periments (9 animals). D: Effects of cen-
obamate on the kinetic parameters (τfast,
62.4 ± 6.1 ms for cenobamate, n = 5, P < 0.01, Fig. 5D), and induced
a statistically significant decrease in Afast but not in Aslow (Afast;
0.66 ± 0.01 for control and 0.55 ± 0.01 ms for cenobamate, n = 5,
P < 0.01, Aslow; 0.36 ± 0.01 for control and 0.38 ± 0.12 ms for
cenobamate, n = 5, P = 0.26, respectively, Fig. 5D).
3.5. Effect of cenobamate on the excitability of CA3 neurons
Persistent Na+ currents are closely related to the basal neuronal
excitability, including resting membrane potentials and firing patterns
(Stafstrom, 2007). Therefore, we next examined the effect of cen-
obamate on the excitability of CA3 pyramidal neurons in a current-
clamp mode. In current-clamp conditions, resting membrane potentials
were −60.3 ± 2.1 mV (n = 9). Application of cenobamate hyperpo-
larized membrane potentials in a concentration-dependent manner with
an EC50 value of 89.8 ± 12.3 μM (n = 7, Fig. 6Aa and Ba). At a10 μM
concentration, cenobamate elicited a hyperpolarization of
1.5 ± 0.3 mV (n = 7, P < 0.01). The cenobamate-induced hyperpo-
larization completely disappeared in the presence of either 10 μM ri-
luzole (−5.6 ± 0.5 mV and −0.2 ± 0.1 mV in the absence and pre-
sence of riluzole, n = 6, P < 0.01) or 300 nM tetrodotoxin
(−5.3 ± 0.4 mV and −0.1 ± 0.1 mV in the absence and presence of
tetrodotoxin, n = 6, P < 0.01) (Fig. 6Ab and Bb). In addition, riluzole
(10 μM) and tetrodotoxin (300 nM) by themselves hyperpolarized
membrane potentials to −7.6 ± 1.1 mV (n = 6) and −11.4 ± 0.8 mV
(n = 6), respectively (Fig. 6Ab). The addition of either riluzole (10 μM)
or tetrodotoxin (300 nM) immediately after the application of cen-
obamate (100 μM) further hyperpolarized membrane potentials
(Fig. 6Ac). These results suggest the riluzole-sensitive INaP is involved in
the excitability of CA3 pyramidal neurons, and that cenobamate can
reduce neuronal excitability by inhibiting the INaP. On the other hand,
cenobamate had no effect on voltage-gated Ca+ or K+ channels
(Supplementary Fig. S2) or excitatory receptors, such as α-amino-3-
hydroxy-5-methylisoxazole-4-propionate (AMPA), kainic acid (KA),
and N-methyl-D-aspartate (NMDA) receptors (Supplementary Fig. S3).
The effect of cenobamate was further examined on the firing pat-
terns in response to a depolarizing current injection (Fig. 6C and D).
While cenobamate (100 μM) significantly increased the rheobase cur-
rents (65.3 ± 15.1 pA for control and 130.6 ± 19.9 pA for cen-
obamate, n = 9, P < 0.01, Fig. 6Da), the input resistance was not
changed by 100 μM cenobamate (251.1 ± 49.9 MΩ for control and
M. Nakamura, et al. European Journal of Pharmacology 855 (2019) 175–182

Fig. 5. Effect of cenobamate on the re-

covery from inactivation of voltage-gated
Na+ channels in hippocampal CA3 neurons.
A: A schematic illustration of voltage step
pulses. The INa was induced by the two-
pulse protocol, where the first conditioning
pulses (P1; −20 mV depolarization, 500 ms
duration) were followed by the second test
pulses (P2; −20 mV depolarization, 50 ms
duration). The second INa was recovered
with various interpulse intervals of
1–900 ms at a potential of −80 mV. B:
Typical first (P1) and second (P2) traces of
INa induced by the two-pulse protocol in the
absence (left) and presence (right) of
100 μM cenobamate. C: Kinetics for the re-
covery from inactivation of voltage-gated
Na+ channels in the absence (open circles)
and presence (closed circles) of 100 μM
cenobamate. Each point is the P2/P1 ratio of
INa induced by two depolarizing step pulses
and represents the mean ± S.E.M. from 5 experiments (5 animals). D: Effects of cenobamate on the kinetic parameters (τfast, τslow, Afast, Aslow, and τweighted) for the
recovery from inactivation of voltage-gated Na+ channels. Each column represents the mean ± S.E.M. from 5 experiments (5 animals). **P < 0.01; n.s., not
significant.

295.7 ± 49.2 MΩ for cenobamate, n = 9, P = 0.08, Fig. 6Db). Cen-
obamate (100 μM) also decreased the number of action potentials
generated by depolarizing current stimuli (Fig. 6Dc).
4. Discussion
In this study, the investigational AED cenobamate did not sig-
nificantly alter the peak amplitude, onset time, or decay time constant
of INaT, indicating that cenobamate had little effect on the transient
component of INa. However, cenobamate did potently inhibit the per-
sistent component of INa. We also found that cenobamate potently in-
hibited slow voltage-ramp-induced current in a concentration-depen-
dent manner, with an IC50 value of 53.1 μM. This IC50 value was similar
to that for the inhibition of INaP, suggesting that INaP might be com-
parable to the slow voltage-ramp-induced current. The much higher
IC50 value (> 500 μM) associated with inhibition of the INaT would

Fig. 6. Effects of cenobamate on the excitability of acutely isolated CA3 pyramidal neurons. Aa: Typical traces of voltage responses during the application of various
concentrations of cenobamate (3 μM–300 μM). Ab: Typical traces of voltage responses during the application of 100 μM cenobamate in the absence (left) and
presence of 10 μM riluzole (middle) or 300 nM TTX (right). TTX, tetrodotoxin. Ac: Typical traces of voltage responses during the consecutive application of 100 μM
cenobamate (C) and 10 μM riluzole (R) or 300 nM TTX (T). Ba: Concentration-response relationships of cenobamate (open circles) against the membrane hy-
perpolarization. The voltage of maximal hyperpolarization for cenobamate was set to 11.4 mV, the same value at which TTX induced membrane hyperpolarization.
Each point represents the mean ± S.E.M. from 4 to 8 experiments (8 animals). Note that either 10 μM riluzole (closed circle) or 300 nM TTX (closed rectangle)
greatly induced membrane hyperpolarization, as also shown in Ab. TTX, tetrodotoxin; Rilu, riluzole. Bb: Cenobamate (100 μM)-induced membrane hyperpolarization
in the absence and presence of 10 μM riluzole or 300 nM TTX. Each column represents the mean ± S.E.M. from 6 experiments (6 animals). **P < 0.01, n.s.; not
significant; TTX, tetrodotoxin. Note that cenobamate did not induce any membrane hyperpolarization after the blockade of INaP by riluzole or TTX. C: Typical traces
of voltage responses during depolarizing current injection in the absence (upper) and presence (lower) of 100 μM cenobamate. D: Effects of cenobamate (100 μM) on
the rheobase currents (a) and input resistance (Rinput, b). Each column represents the mean ± S.E.M. from 9 experiments (9 animals). **P < 0.01; CNB, cen-
obamate; Cont, control; n.s., not significant. Dc: Effect of cenobamate (100 μM) on the number of action potentials elicited by each depolarizing current injection
(1T–4T). T indicates the amplitude of minimal current to elicit action potentials. Each point represents the mean ± S.E.M. from 9 experiments.

180
M. Nakamura, et al.
suggest that cenobamate may preferentially inhibit the INaP.
Furthermore, cenobamate showed a higher preference for the INaP in-
hibition compared to either carbamazepine or lamotrigine; this may
contribute to the relative differences in efficacy and tolerability among
these known agents. In addition, under the concentrations examined in
these analyses, cenobamate would be expected to significantly inhibit
the persistent component of INa at clinically therapeutic doses
(equivalent to doses ranging from 100 to 400 mg/day).
As shown in this study, slow voltage-ramp-induced membrane
currents were recorded from the majority of acutely isolated CA3 pyr-
amidal neurons from rats. The major portion of these currents appeared
to be INaP-mediated by voltage-gated Na+ channels, as they were al-
most completely blocked either by tetrodotoxin, a specific voltage-
gated Na+ channel blocker, or riluzole, which is known to inhibit INaP
(Urbani and Belluzzi, 2000). On the other hand, either riluzole or te-
trodotoxin by itself induced membrane hyperpolarization in current-
clamp experiments, suggesting that tonic INaP might participate in the
regulation of resting membrane potentials of acutely isolated CA3
pyramidal neurons. Although the tetrodotoxin-induced membrane hy-
perpolarization in the same neuron was found in our previous study
(Jang et al., 2006), further examination would be needed to elucidate
the detailed mechanism underlying the involvement of tonic INaP in the
regulation of resting membrane potentials.
As many commercially available AEDs affect the voltage depen-
dence of Na+ channels (Catterall, 2014; Macdonald and Kelly, 1995;
Spadoni et al., 2002), we also examined the effects of cenobamate on
the voltage-dependent activation and inactivation of Na+ channels. We
found that changes in V50,act values induced by cenobamate were not as
prominent as the dose-dependent changes in V50,inact values, suggesting
that the effect of cenobamate on Na+ channels is mainly mediated by
modulating the voltage-dependent fast inactivation rather than acti-
vation. Since cenobamate had no inhibitory effect on the INa at hy-
perpolarized membrane potentials, a negative shift of steady-state fast
inactivation relationship might be responsible for the cenobamate-in-
duced decrease in transient INa at −80 mV. In addition, the cenobamate
inhibition of peak INa of voltage-gated Na+ channels was more potent at
depolarized membrane potentials, indicating that cenobamate has a
much higher affinity for the inactivated than for resting Na+ channels.
This was further supported by the KI and KR values (48.2 μM and
797.8 μM, respectively). As a number of other Na+ channel blocking
AEDs are state-dependent and have a higher affinity for inactivated
Na+ channels, a higher affinity of cenobamate for the inactivated
channels than for resting Na+ channels would be expected to contribute
to its anti-epileptic effects (Macdonald and Kelly, 1995). The kinetics
regarding the inactivation property of voltage-gated Na+ channels,
such as the development of inactivation and recovery from fast in-
activation, are essential for excessive neuronal excitability, such as re-
petitive and bursting generation of action potentials. In the present
study, cenobamate significantly accelerated the overall inactivation
kinetics, and the extent of this acceleration was higher in the fast (τfast)
than in the slow component (τslow). Assuming that the two time con-
stants reflect the extent of fast and slow inactivation, respectively,
cenobamate might preferentially accelerate the fast inactivation during
sustained membrane depolarization. In addition, we found that cen-
obamate significantly decreased the overall recovery kinetics, and the
extent of the decrease was greater in the τslow than in the τfast, in-
dicating that cenobamate would further retard the recovery from slow
inactivation. In this stage, the mechanisms underlying the cenobamate-
induced changes in inactivation kinetics are unknown. However, given
that the INaP is a non-inactivating component of INa during sustained
membrane depolarization, and that cenobamate preferentially inhibits
the INaP than the INaT, the suppression of INaP by cenobamate might
result in the change of inactivation and/or recovery kinetics of Na+
channels.
Despite the small amplitude, the INaP, in particular at subthreshold
membrane potentials, plays pivotal roles in neuronal excitability,
181
European Journal of Pharmacology 855 (2019) 175–182
resulting in the facilitation of repetitive firing (Stafstrom, 2007). In
pathological conditions, evidence suggests that the INaP is closely re-
lated to epilepsy. For example, point mutations in the sodium channel,
voltage-gated alpha subunit NaV1.1, such as T875M, W1204R, and
R1648H, show impaired inactivation of Na+ channels, resulting in
generalized epilepsy with febrile seizures plus (GEFS+) (Lossin et al.,
2002). In addition, mutations in NaV1.2 (Scn2aQ54) also lead to im-
paired inactivation of Na+ channels and are associated with severe
epileptic behaviors (Bergren et al., 2005; Kearney et al., 2001). These
mutant Na+ channels also show increased non-inactivating INa and/or
INaP, suggesting possible roles of INaP in the pathophysiology of epilepsy
(Stafstrom, 2007; Sugawara et al., 2001). Furthermore, increased INaP
in the pilocarpine-induced status epilepticus rat model of temporal lobe
epilepsy contributed to establishing chronic epilepsy in this rodent
model (Chen et al., 2011). A recent study has shown that preferential
INaP blockade has anti-epileptic effects in Scn2aQ54 animals (Anderson
et al., 2014). In fact, Na+ channel blockers that are used to treat epi-
lepsy, such as phenytoin, valproic acid, carbamazepine, and lamo-
trigine, are also known to block the INaP to some degree (Kohling, 2002;
Niespodziany et al., 2004; Spadoni et al., 2002). Given that the INaP
blockade underlies the anti-epileptic effect mediated by cenobamate,
further investigation is needed to determine whether cenobamate in-
hibits the INaP from mutated Na+ channels. In addition, it would be
worth evaluating the clinical effect of cenobamate on epileptic patients
having mutated Na+ channels.
In current-clamp experiments, cenobamate hyperpolarized
membrane potentials in a concentration-dependent manner,
reaching statistical significance at ≥10 μM cenobamate. The cen-
obamate-induced hyperpolarization was completely blocked either
by tetrodotoxin or riluzole, suggesting the involvement of INaP in
such hyperpolarization. A hyperpolarization of membrane potentials
can be mediated by the inhibition of excitatory receptors such as
AMPA, KA and NMDA receptors. However, these receptors were not
likely to be involved in the cenobamate-induced hyperpolarization,
because cenobamate at a 100 μM concentration did not affect other
voltage-gated ion channels and/or ionotropic glutamate receptors
that are able to induce membrane hyperpolarization (see
Supplementary Figs. S2 and S3). We also found that cenobamate
increased the rheobase currents rather than input resistance in CA3
pyramidal neurons. Considering the minimal effect on the transient
INa, the cenobamate-induced membrane hyperpolarization might be
responsible for the increase in threshold for the generation of action
potentials. More importantly, cenobamate reduced the number of
action potentials triggered by depolarizing current stimuli. Given
that the INaP is involved in the repetitive and burst generation of
action potentials in central neurons (Wu et al., 2005; Yue et al.,
2005), the preferential blockade of INaP by cenobamate may con-
tribute to its anti-epileptic activity against bursting neurons.
In addition to the INaP, hyperpolarization-gated and cyclic nu-
cleotide-activated cation (HCN) currents mediated by HCN channels
are also involved in the repetitive and rhythmic generation of action
potentials in various central neurons (Biel et al., 2009; Khaliq and
Bean, 2010; Puopolo et al., 2007). In the present study, we could not
record any HCN channel-mediated voltage responses in acutely iso-
lated CA3 pyramidal neurons. Furthermore, cenobamate did not af-
fect the voltage changes in response to hyperpolarizing current sti-
muli, suggesting that the INaP rather than HCN channel-mediated
currents is a pharmacologic target for cenobamate. In fact, HCN
channel inhibitors do not show effects on the firing pattern in several
neurons that display repetitive firing activity (Khaliq and Bean,
2010; Taddese and Bean, 2002).
In conclusion, we have examined the effects of cenobamate, a newly
developed AED, on neuronal voltage-gated Na+ channels. In addition to
its modulation of several properties of voltage-gated Na+ channels,
cenobamate acts as a preferential INaP inhibitor in neuronal voltage-
gated Na+ channels to exert its anti-epileptic efficacy.
M. Nakamura, et al.
Conflicts of interest
MN: Declarations of interest: none.
JHC: Declarations of interest: none.
HS: Employee of SK Biopharmaceuticals.
ISJ: Declarations of interest: none.
Authors’ contributions
MN carried out the electrophysiological studies and drafted/criti-
cally reviewed the manuscript.
JHC carried out the electrophysiological studies.
HS participated in designing the study and critically reviewed the
manuscript.
ISJ participated in designing the study, carried out the electro-
physiological studies, and drafted/critically reviewed the manuscript.
All authors read and approved the final manuscript.
Prior presentation/publication
Nakamura M et al. Mechanism of action of cenobamate: preferential
inhibition of the persistent sodium current. Presented at the 2018
Annual Meeting of the American Academy of Neurology, April 21–27,
Los Angeles, CA. Poster P5.278.
Funding & role of sponsor
This study was supported by SK Biopharmaceuticals Co., Ltd.
Medical writing support was funded by SK Life Science, Inc. As an
employee of the study sponsor, HS participated in designing the study,
critically reviewed the manuscript, and read and approved the final
manuscript.
Acknowledgments
The authors thank Sarah Mizne, PharmD, and Miriam Gitler, PhD, of
MedVal Scientific Information Services, LLC, for providing medical
writing and editorial support, which were funded by SK Life Science,
Inc. This manuscript was prepared according to the International
Society for Medical Publication Professionals' “Good Publication
Practice for Communicating Company-Sponsored Medical Research:
GPP3.”
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejphar.2019.05.007.
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