Pharmacological Reports
https://doi.org/10.1007/s43440-023-00477-1

SHORT COMMUNICATION

Inhibitory effect of atomoxetine on Nav1.2 voltage‑gated sodium
channel currents
Yoshihiko Nakatani1· Kanami Ishikawa1 · Yuko Aoki1 · Takahiro Shimooki1 · Naoki Yamamoto1,2,3 · Taku Amano4

Received: 18 December 2022 / Revised: 3 March 2023 / Accepted: 5 March 2023
© The Author(s) under exclusive licence to Maj Institute of Pharmacology Polish Academy of Sciences 2023

Abstract
Background Atomoxetine (ATX), a norepinephrine reuptake inhibitor (NRI), is used to attenuate the symptoms of Attention Deficit/Hyperactivity Disorder (AD/HD) by increasing neurotransmitter concentrations at the synaptic cleft. Although
Nav1.2 voltage-gated sodium channels (VGSCs) are thought to play a role in monoamine transmitter release in the synaptic
junction, it is unclear how atomoxetine affects Nav1.2 VGSCs.
Methods In this study, we investigated the effect of ATX on Nav1.2 VGSC-transfected HEK293 cells with the whole-patch
clamp technique.
Results Nav1.2 VGSC current decreased by 51.15 ± 12.75% under treatment with 50 µM ATX in the resting state (holding
membrane potential at – 80 mV). The ­IC50 of ATX against Nav1.2 VGSC current was 45.57 µM. The activation/inactivation
curve of Nav1.2 VGSC currents was shifted toward hyperpolarization by 50 µM ATX. In addition, the inhibitory effect of
ATX increased with membrane depolarization (holding membrane potential at – 50 mV) and its I­ C50 was 10.16 µM. Moreover, ATX showed the time-dependent interaction in the inactivation state.
Conclusion These findings suggest that ATX interacts with Nav1.2 VGSCs producing the inhibition of current and themodification of kinetic properties in the state-dependent manner.
Keywords Atomoxetine · HEK293 · Nav1.2 VGSCs
Abbreviations
NRI	Norepinephrine reuptake inhibitor
AD/HD	Attention deficit/hyperactivity disorder
VGSCs	Voltage-gated sodium channels
NET	Norepinephrine transporter
DAT	Dopamine transporter
ATX	Atomoxetine* Yoshihiko Nakatani
ynakatani@iuhw.ac.jp
1

Department of Pharmacotherapeutics, School of Pharmacy,
International University of Health and Welfare, 2600‑1
Kitakanemaru, Ohtawara, Tochigi 324‑8501, Japan

2

Center for Basic Medical Research, International University
of Health and Welfare, 2600‑1 Kitakanemaru, Ohtawara,
Tochigi 324‑8501, Japan

3

Laboratory of Neurobiology, Tokyo Metropolitan University,
1‑1 Minami‑Osawa, Hachioji‑Shi, Tokyo 192‑0397, Japan

4

Tochigi Prefectural Okamotodai Hospital, 2162
Shimookamotomachi, Utsunomiya, Tochigi 329‑1104, Japan

Introduction
Atomoxetine (ATX) is the first non-stimulant antipsychotic
drug approved for the treatment of Attention Deficit/Hyperactivity Disorder (AD/HD) in adults [1]. ATX is consideredto improve various symptoms in AD/HD, such as impairments of modulating attention, working memory, behavioral inhibition, planning alertness, arousal, and vigilance, byincreasing the concentration of norepinephrine at the synaptic cleft though inhibiting the function of norepinephrinetransporters (NET) in the prefrontal cortex. Moreover, there
is no risk of addiction with ATX since it does not block
dopamine transporters (DAT) of the reward system in thesubstantia nigra pars compacta and ventral tegmental area[2]. Thus, ATX shows a high affinity for NET with a K­ ivalue of 5 nM, whereas its affinity for the dopamine transporter (DAT) is much lower ­(Ki value for DAT: 1450 nM)[3]. However, since typical plasma concentrations of ATX
at the time of therapeutic treatment (1–3 µM) are high compared to ­Ki values of ATX to NET or DAT calculated fromin vitro experiments [4], the interaction of ATX at the time

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Y. Nakatani et al.

of therapeutic treatment may not be restricted to NET and
DAT alone.
Nav1.2 voltage-gated sodium channels (VGSCs), which
are highly expressed in neurons in the brain, contributeaction potential to initiation [5]. Thus, a change, such as
gain-of-function or loss-of-function, of Nav1.2 may leadto various symptoms induced by the dysfunction of neuronal excitability in the central nervous system, as typified
by epilepsy [6, 7]. Therefore, it considered that the functional modulation of Nav1.2 VGSCs function may affectthe release of neurotransmitters including norepinephrine.
In addition, it has been reported that ATX interacted with
Nav1.5 VGSCs in a state- and use-dependent manner [8].
According to the studies mentioned above, it is possible that
Nav1.2 may be an as-yet unknown target for ATX in thetherapeutic condition in which its plasma concentration rises
above the ­Ki value of NET.
In this study, we investigated the effect of ATX on theNav1.2 VGSCs using HEK293 cells transfected with the rat
SCN2A gene encoding Nav1.2 VGSCs.

Materials and methods
Establishment and culture of Nav1.2 VGSCs
expressing HEK293 cells
The establishment of Nav1.2-expressing HEK293 cells andthe culture protocol have been described previously [9]. In
brief, the cDNA of Nav1.2 VGSCs inserted into pCI-neo
Mammalian Expression Vector (Promega, Fitchburg, WI,
U.S.A.) was transfected with FuGENE® 6 Transfection Reagent (Promega) into HEK293 cells (JCRB Cell Bank, Osaka,
Japan). Cells expressing Nav1.2 VGSCs were then selectedwith media containing 500 µg/mL Geneticin (Nacalai
Tesque, Inc., Kyoto, Japan). In general, cells were passaged
once every 5–7 days.

Electrophysiology
Whole-cell VGSC currents were recorded as described
previously with some slight modifications [9]. For electrophysiological experiments, cells that had been cultured
1–4 days from passage on poly-L-lysine-coated cover glass(AGC Techno Glass Co., Ltd., Shizuoka, Japan) were used.
In general, culture media containing 500 µg/mL Geneticin
was changed every other day to maintain the cell condition. All experiments were performed at 22–25 °C in abath solution containing (mM) 140 NaCl, 0.03 ­CaCl2, 10
HEPES, 10 M
­ gCl2·6H2O and 10 d-glucose, and the pH wasadjusted to 7.4 with NaOH. The microelectrode solution
consisted of (mM) 115 ­CsCl2, 25 NaCl, 2 ­MgCl2·6H2O, 1­CaCl2, 11 EGTA and 10 HEPES, and the pH was adjusted

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to 7.4 with CsOH. The patch microelectrodes for recording were made from borosilicate capillary glass (Narishige,
Tokyo, Japan) and had resistances of 3–5 MΩ. Whole-cellcurrents were recorded at 50 kHz after low-pass filtering
at 3 kHz using EPC-7 Plus and EPC-8 amplifiers (HEKA
Elektronik, Lambrecht, Germany) and digitized with Digidater1322A and 1440A (Molecular Devices, Sunnyvale, CA,
USA). Leak currents were subtracted by a P/4 pulse protocol. These recorded data were sampled and analyzed using
pCLMAP10.6 software (Molecular Devices).
ATX was purchased (Tokyo Chemical Industry Co., Ltd.,Tokyo, Japan) and dissolved in DMSO as a stock solution.
The final concentration of DMSO in the bath solution was
set at 0.1%. To explore the effect of ATX on Nav1.2 VGSC
currents, the bath was perfused with a bath solution in which
ATX was dissolved at the indicated concentrations.

Data analysis
Data were analyzed using a combination of pCLAMP 10.6
software, Origin 6.1, Microsoft Excel, and GraphPad InStat
3 software. The results are presented as the mean ± SD. Fora single comparison of paired data, a statistical analysis was
performed using paired t-test. A p-value < 0.05 was considered to be statistically significant.

Results
Interaction of atomoxetine with the resting state
First, we investigated the effect of atomoxetine on Nav1.2
VGSCs peak current in the resting state (holding membrane potential at – 80 mV). Figure 1A shows the effectof 50 μM ATX on the Nav1.2 VGSC peak current evoked
by depolarization from a holding potential of − 80 mV
to a testing potential of 10 mV in HEK293 cells. Under
treatment with 50 μM ATX, the Nav1.2 VGSC peak current decreased by around 50% compared to those before
treatment with ATX (Fig. 1A). After ATX was washedout, the Nav1.2 VGSC peak current that had been inhibited by the treatment with ATX was almost restored. Figure 1B shows the current-voltage relationship before andduring treatment with 50 µM ATX and after washing out.
The inhibitory effect of 50 μM ATX started at around− 20 mV and reached a maximum at 10 mV, after which
inhibition persisted with further depolarization potentials (Fig. 1B). Nav1.2 peak VGSC current was decreased
by 51.15 ± 12.75% under treatment with 50 μM ATX(Fig. 1B) compared to before treatment. The time coursechanges of Nav1.2 VGSCs peak current inhibitory effectof 50 µM ATX and reversibility (washing out with bathsolution) are shown in Fig. 1C. 50 µM ATX inhibited theInhibitory effect of atomoxetine on Nav1.2 voltage‑gated sodium channel currents﻿	Fig. 2  Concentration-dependent effect of ATX on Nav1.2 VGSCs
peak current. Each point on the concentration-response curve represents the mean ± SD (10 μM; n = 7, 30 μM; n = 4, 50 μM;   n = 10,85 μM; n = 4, 100 μM; n = 4, 300 µM; n = 4). The inhibition ratioof ATX at each concentration was fitted using a logistic function;
A2 + (A1-A2)/1 + (X-Xh)p. A1 and A2 represent the initial value andfinal value, respectively and X and Xh are the actual concentrationand the concentration at which half-maximal inhibition is shown. I­ C50
was determined to be 45.57 µM

Effect of atomoxetine in the activationand inactivation
Fig. 1  Effect of ATX on Nav1.2 VGSCs current. (A) Current traces
recorded from rat Nav1.2 VGSCs expressed HEK293 cells before andafter treatment with 50 µM ATX and after washing out of ATX. To
record Nav1.2 VGSCs current, cells were held at – 80 mV as holding
potential and stepped to a test pulse at 10 mV for 20 ms. (B) Current-voltage relationship of normalized Nav1.2 VGSCs current. The
normalized peak Nav1.2 VGSCs current before treatment (open circle), under treatment with 50 µM ATX (filled circle) and after washing out of ATX (filled square) were plotted against each depolarizing
test potential. Each current-voltage relationship curve is shown as themean ± SD (n = 6). (C) The time course changes of the normalized
current amplitude of Nav1.2 VGSCs peak current before and during
treatment with 50 µM atomoxetine and after washing out of atomoxetine. The line graph shows mean ± SD (n = 6)

Nav1.2 VGSCs peak current gradually, and this inhibition was reversed by washing out ATX with bath solution(Fig. 1C).
Figure 2 shows the concentration-dependent inhibitory
effect of ATX on Nav1.2 VGSC peak currents. The inhibition ratio of ATX at each concentration was fitted using
a logistic function. Treatment with 10, 30, 50, 85, 100
or 300 µM ATX decreased the Nav1.2 VGSC peak currents by 1.96 ± 16.37%, 14.56 ± 7.57%, 51.15 ± 12.75%,
76.41 ± 11.76%, 68.72 ± 8.05 and 87.72 ± 8.10%, compared to before treatment with ATX, respectively. Whenthe inhibitory effect of Nav1.2 VGSCs peak currents
obtained under the treatment with 300 µM ATX was taken
to represent as a maximal inhibitory response, ­IC50 was
determined to be 45.57 µM.

Figure 3A shows the effects of ATX on the activation
curves of Nav1.2 VGSCs. To record Nav1.2 VGSC activation current, the cell was held at − 80 mV and stepped
to a depolarizing pulse (from − 100 to 30 mV) for 20 ms.
The voltage dependence of activation was calculated in two
steps. First, changes in driving force in the different test
potentials were calculated by the conductance G according
to G = I(E – Erev). Then, normalized conductance was plotted against the potential of the depolarizing pulse and fittedwith the Boltzmann function. The voltage at half-maximal
activation changed significantly from − 5.05 ± 3.08 mV to− 9.38 ± 3.30 mV under the treatment with 50 µM ATX(Fig. 3B, n = 6, ­t5 = 5.25, p = 0.0033). Figure 3C shows theeffects of ATX on the inactivation curves of Nav1.2 VGSCs.
To record Nav1.2 VGSCs inactivation current, cells were
held at − 80 mV and stepped to an inactivation pulse (from– 100 to 20 mV) for 1 s in steps of 10 mV. Inactivation wasdetermined by plotting the normalized Nav1.2 VGSCs peakcurrent during the following test pulse at – 10 mV for 20 ms
versus the pre-pulse potential. The voltage at half-maximal
inactivation also changed slightly but significantly from– 50.58 ± 3.18 mV to – 52.64 ± 4.43 mV under the treatment
with 50 µM ATX (Fig. 3D, n = 15, ­t14 = 3.38, p = 0.0045).

Interaction of atomoxetine with the inactivating
state
Since the inhibitory effect of ATX on Nav1.2 VGSCs peakcurrent in the resting state was relatively small, we assessed13

Y. Nakatani et al.

Fig. 3  Effects of ATX on the voltage-dependence of activation andinactivation of Nav1.2 VGSCs. (A) Plots of normalized peak conductance (G/Gmax). These data sets, before (open circle) and after
treatment with 50 µM ATX (filled circle), were fitted with a Boltzmann function; 1/1 + exp((V−Vh)/k). V and Vh are the actual membrane potential and the potentials at which half-maximal current or
conductance are shown. Each activation curve (A) and half activation potential (B) represent the mean ± SD (n = 6), respectively. To
analyze the difference of V
­ 1/2 in the activation curve between before
treatment and 50 µM ATX, a paired t-test was performed. (C) Plots ofnormalized inactivation peak currents. These data sets, before (opencircle) and after treatment with 50 µM ATX (filled circle), were fittedwith a Boltzmann function. Each inactivation curve (C) and half inactivation potential (D) represent the mean ± SD (n = 15), respectively.
To analyze the difference of ­V1/2 in the inactivation curve before
treatment and 50 µM ATX, a paired t-test was performed

the effect of ATX on Nav1.2 VGSCs peak current in the inactivated state. The holding membrane potential was chosen
to be – 50 mV which is considered the threshold membranepotential in the neuron. Figure 4A shows the concentrationdependent inhibitory effect of ATX on Nav1.2 VGSC peakcurrents. The inhibition ratio of ATX at each concentration
was fitted using a logistic function. Treatment with 0.5, 1,
5, 10, 30 or 50 µM ATX decreased the Nav1.2 VGSC peakcurrents by – 8.09 ± 5.86%, 5.04 ± 15.07%, 27.59 ± 17.78%,
42.78 ± 14.70%, 81.34 ± 7.71 and 83.64 ± 10.99%, compared
to before treatment with ATX, respectively. When the inhibitory effect of Nav1.2 VGSCs peak currents obtained under
the treatment with 50 µM ATX was taken to represent a
maximal inhibitory response, I­ C50 was determined to be
10.16 µM.

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Fig. 4  Effects of ATX on the inactivated state of Nav1.2 VGSCs. (A)Concentration-dependent effect of ATX on Nav1.2 VGSCs peak current with holding potential at – 50 mV. Each point on the concentration-response curve represents the mean ± SD (0.5 μM; n = 4, 1 μM;
n = 4, 5 μM; n = 5, 10 μM; n = 4, 30 μM; n = 4, 50 µM; n = 4). ­IC50
was determined to be 10.16 µM. (B) Time-dependent effect of ATX
on Nav1.2 VGSC inactivated state. The peak currents of the test pulse
were normalized with the peak currents of conditioning pre-pulse forinactivation and were plotted vs. the duration of conditioning prepulse in the absence of ATX (control; open circle), in the presenceof 10 µM ATX (filled triangle) and 50 µM ATX (filled circle). These
data sets were fitted with single exponential functions. Each point
represents the mean ± SD (control; n = 9, 10 μM; n = 5, 50 μM; n = 8)

As shown in Fig. 4B, we examined the effect of ATX in
the time-dependence interaction with the inactivation state.
To assess the time dependence interaction, cells were heldat – 80 mV and stepped to an inactivation pulse at 10 mV
lasting 10 ms to 5120 ms, followed by a short recovery for
100 ms at – 80 mV and ending with a 20 ms test pulse at
10 mV. The pulse protocol mentioned above was appliedin an interval of 20 s. In the absence of ATX, the control
values decreased after inactivation pulses lasting longer than
100 ms and dropped to 36.0% ± 14.16% of the conditioning pre-pulse amplitude after the longest inactivation pulseof 5120 ms. In the presence of 10 µM ATX, the decreaseof the relative current amplitude of the test pulse to conditioning pre-pulse started in a shorter duration of conditioning pre-pulse with stronger than the absence of ATX(remaining current was 7.57% ± 4.56% at 5120 ms). With theInhibitory effect of atomoxetine on Nav1.2 voltage‑gated sodium channel currents﻿	higher concentration of ATX (50 µM), the inhibitory effectemerged even when conditioning pre-pulse was applied for
10 ms (remaining current: 79.49% ± 5.53%), and the relative
current amplitude of conditioning pre-pulse at the longest
inactivation time (5120 ms) dropped to – 0.53% ± 4.08%(Fig. 4B).
Tonic and use‑dependent block of atomoxetine
To assess the interaction of ATX with the open channel, we examined whether ATX showed a use-dependent
block or not. After establishing the baseline condition in
the absence of ATX in each cell with the test pulse, bathsolution was exchanged to it containing 50 µM ATX andthen the test pulse was employed to the cells to activate
every 5 s. For evaluation, the peak current amplitude ofeach pulse was divided by the average of the peak current
amplitude obtained from the baseline condition and plottedvs. pulse number. All experiments were performed at theholding potential of − 80 mV and the test pulse was 10 mV
for 20 ms. In the absence of 50 µM ATX, the use-dependent
behavior was not observed (Fig. 5). When the test pulse was
employed to the cells that were pre-treated with 50 µM ATX,
it was observed the tonic block by the treatment with 50 µ
ATX and the relative current amplitude was decreased to78.15 ± 4.09% in the first activation with test pulse (Fig. 5).
After the test pulse was employed to the cells continuously,
the moderate use-dependent block was observed and relative
current amplitude was decreased to 59.30 ± 4.41% finally(Fig. 5).

Fig. 5  Use-dependent interaction of ATX on Nav1.2 VGSCs. Afterestablishing baseline current amplitude response, bath solution was
exchanged to it containing 50 µM ATX and pre-incubated before thetest pulse was applied to the cells. Under control condition (open circle), use-dependence was not observed. In contrast, both tonic anduse-dependent blocks were observed in the presence of 50 µM ATX(filled circle). Each point represents the mean ± SD (control; n = 4,
50 μM; n = 4)

Discussion
The present study was designed to update the pharmacological information on ATX which might affect othermolecules in the brain other than NET. Our study demonstrated that ATX inhibited resting state Nav1.2 VGSC peakcurrent amplitude in a concentration-dependent manner. In
addition, we also clarified that ATX shifted both the activation and inactivation curves of Nav1.2 VGSCs toward
hyperpolarization in the resting state. Moreover, when
we examined the effect of ATX in the inactivated state ofNav1.2 VGSCs, we found that ATX intensified inhibitionof Nav1.2 VGSC peak current amplitude compared to theresting state, and its inhibition was correlated with thelength of inactivated duration. In addition, ATX induced
both tonic and use-dependent blocks on the Nav1.2 VGSCs
peak current amplitude.
A recent study demonstrated that ATX also inhibited Nav1.5 VGSC currents, which are predominantly
expressed in the myocardium, with a relatively low concentration at 3 µM [8]. Considering that the calculated­I C 50 of ATX for the Nav1.2 VGSC peak current in ourstudy was 45.57 µM in the resting state, and 10.16 µM in
the inactivated state (the holding potential at – 50 mV),it might be concluded that the affinity of ATX for Nav1.5
VGSCs is greater than that for Nav1.2 VGSCs. However, this inhibitory effect of ATX on Nav1.5 VGSC currents was observed when the holding potential was set at– 85 mV, which is approximately the half-maximal inactivated condition. Indeed, the same study also reported that
3 µM ATX could decrease Nav1.5 VGSC currents by only
12.8% and the half-maximal inhibitory effect of ATX on
Nav1.5 VGSC currents was 119 µM in the resting state
when the holding potential was set at – 140 mV [8]. Considering that ATX showed both tonic and use-dependent
block on Nav1.2 VGSCs current amplitude, the inhibitory
effect of ATX on Nav1.2 VGSCs may be more potent thanthat of ATX for Nav1.5 VGSCs in the resting state.
It has been reported that the plasma concentrationlevel of ATX in therapeutic dose application ranges from
1–3 µM [10]. However, the concentration of ATX in brain
tissue after single or repeated treatment is not known yet.
Considering that the plasma concentration of ATX tends
to elevate after chronic treatment in some patients [4, 11]
and the I­ C50 of ATX for Nav1.2 VGSCs in the inactivatedstate was around 10 µM and its value is not so far fromthe plasma concentration range of ATX at the therapeutic
condition, it is possible that accumulated ATX inhibits
Nav1.2 VGSCs in a state-dependent manner. In this study,
we also demonstrated that both the voltage dependence ofactivation and inactivation were shifted slightly toward
the hyperpolarization side in the resting state. However,13

Y. Nakatani et al.

these changes may be too little to affect to the physiological aspect at the therapeutic plasma concentration of ATX.
The primary target molecule of ATX through which it
exhibits its therapeutic effect in the treatment of AD/HD isconsidered to be NET, not Nav1.2 VGSCs. Considering that
Nav1.2 VGSCs contribute to initiating an action potential,
though we first expected that ATX activated Nav1.2 VGSCs,
we found that ATX inhibited Nav1.2 VGSCs activation in
fact. However, the possibility that inhibition of Nav1.2
VGSCs may contribute to the therapeutic effect of ATX
is raised when we integrate the results from several studies. An association between increased pyrethroid pesticide
exposure, which might cause abnormalities in the dopamine
system, and AD/HD in US children has been reported [12].
Deltamethrin, a pyrethroid pesticide, was shown to prolong
sodium channel current in mouse neuroblastoma N1E-115
cells expressing Nav1.2 VGSCs [13, 14]. In addition, singlechannel analysis demonstrated that deltamethrin increased
the population of long-duration openings, consequently
increasing ­Na+ selectivity in rat Nav1.2 VGSCs expressedin Xenopus oocytes [15]. Considering these results, it ispossible that the activation of VGSCs including Nav1.2
expressed in the central nervous system may be related tothe expression of AD/HD symptoms; therefore, the inhibition of Nav1.2 VGSCs by ATX might partially contribute toattenuating symptoms of AD/HD under certain conditions.
There is an apparent contradiction because the inhibition of Nav1.2 VGSC currents induced by ATX theoretically decreases the release of neurotransmitters including
norepinephrine at the synaptic cleft although ATX exerts
its therapeutic effect in the treatment of AD/HD throughan increase in neurotransmitter by inhibiting NET function.
However, it has also been shown that loss-of-function of
Nav1.2 VGSCs causes epilepsy which is considered to be
induced by the excitation of neurons in the brain [6, 16]. In
addition, a recent study demonstrated the down-regulationof multiple potassium channels including Kv1.1 followed byenhanced intrinsic neuronal excitability in Nav1.2 VGSCdeficient neurons; therefore, this mechanism may cause epilepsy under loss-of-function of Nav1.2 VGSCs [17]. It has
been reported that the sodium channel activator, reduced
the expression of sodium channel alpha-subunit mRNA and
its reduction was blocked by the treatment of tetrodotoxin,
a VGSCs blocker, in developing neurons [18]. Thus, theinhibitory effect of ATX on Nav1.2 for AD/HD treatment
is possible to be explained as follows: long-term exposure
to ATX that leads to the prolonged inhibition of Nav1.2
VGSCs may change the expression levels of various ionchannels including itself on neuronal membrane followedby an increase in membrane excitability to enhance neurotransmitter release. Considering that the plasma concentration of ATX in the therapeutic condition is 1–3 µM or
may elevate higher depending on the patient background,13

it is possible that the function of Nav1.2 VGSCs may be
inhibited under certain conditions partially. However, since
it remains whether the chronic pharmacological inhibitionof Nav1.2 VGSCs affects the expression levels of various
types of ion channels in neurons, further examinations will
be needed to clear its possibility.
In summary, ATX inhibited Nav1.2 VGSC peak currents
in a concentration-dependent manner. This inhibition was
more intense in the inactivated state than the resting stateof Nav1.2 VGSCs and its inhibition in the inactivation state
showed in the time-dependent manner. Moreover, both voltage dependence of activation and inactivation were shifted
toward hyperpolarization by treatment with ATX in theresting state. Considering the possible impact of ATX on
Nav1.2 VGSCs in neurons, ATX may decrease the activityof Nav1.2 VGSCs in the central nervous system within thebrain. Further studies will be needed to clarify whether thecorrelation between the electrophysiological mechanismsof the inhibitory effect of atomoxetine on Nav1.2 VGSCs
contributed to its therapeutic effect.
Acknowledgements We specially thank Dr. Weitemier (Tokyo Metropolitan University) gave constructive advice to refine our manuscript.
We also thank the members of the Department of Pharmacotherapeutics, School of Pharmacy, International University of Health and Welfare, for their helpful discussions throughout this study.
Author contribution YN designed this study initially. YN, KI, YA and
TS generated experimental data. YN also analyzed experimental dataand created figures. YN wrote the manuscript with support from NY
and TA.
Funding This work was supported by an internal research grant fromInternational University of Health and Welfare.
Data availability The datasets generated during the current study areavailable from the corresponding author on reasonable request.

Declarations
Conflict of interest None to declare.

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