Functional Modulation of Na_v1.2 Voltage-Gated Sodium Channels Induced by Escitalopram

Abstract

Escitalopram, a selective serotonin reuptake inhibitor (SSRI), may induce seizures, particularly in epileptic patients. In this study, we investigated the effect of escitalopram in Na_v1.2 voltage-gated sodium channels (VGSCs) transfected HEK293 cells. Na_v1.2 VGSCs current decreased by approximately 50.7±8.3% under treatment with 100 µM escitalopram. The IC₅₀ of escitalopram against Na_v1.2 VGSCs was 114.17 µM. Moreover, the treatment with 100 µM escitalopram changed the voltage-dependence of inactivation and the voltage at half-maximal inactivation shifted significantly from −50.3±3.7 to −56.7±6.0 mV toward negative potential under treatment with 100 µM escitalopram. Surprisingly, the treatment with 100 µM escitalopram also changed the voltage-dependence of activation and the voltage at half-maximal activation shifted significantly from −13.8±4.6 to −21.5±3.9 mV toward negative potential under treatment with 100 µM escitalopram. These findings suggested that escitalopram might be able to inhibit Na_v1.2 VGSCs current and affects both activation and inactivation states of Na_v1.2 VGSCs.

Escitalopram is a selective serotonin reuptake inhibitor (SSRI) that was developed as the (S)-stereoisomer of citalopram. Escitalopram is highly selective for serotonin transporters compared to other SSRIs.^1,2) Therefore, both the frequency and severity of the side effects induced by escitalopram are less than those of other SSRIs.^3,4) Nevertheless, some reports have stated that escitalopram is associated with prolongation of the corrected QT (QTc) interval and its effect is related to the blockade of sodium channels, since this prolongation was recovered by treatment with sodium bicarbonate.⁵⁾ In addition, it has been considered that escitalopram, along with other antidepressants, may induce seizures, such as in epileptic patients, though the rate at which it induces seizures is relatively low.^6,7) It is well known that seizures are caused by the unpredictable firing of neurons in the central nervous system related to functional changes in voltage-gated sodium channels (VGSCs) accompanied with several genetic modifications. Among the four types of VGSCs that are predominantly expressed in the central nervous system, i.e., Na_v1.1, Na_v1.2, Na_v1.3 and Na_v1.6, a missense mutation in Na_v1.2 was identified in one patient with generalized epilepsy with febrile seizures and nine patients with benign familial neonatal-infantile seizures.⁸⁾ In particular, three of these nine patients with benign familial neonatal-infantile seizures showed a reduction of sodium channel activity.⁹⁾ In addition, it has been reported that several anticonvulsants, which could show antiepileptic effects by reducing the activities of various VGSCs expressed in the central nervous system, aggravated seizures in a genetic mouse model of epilepsy.¹⁰⁾ According to these studies, escitalopram may have various side effects, such as prolongation of the QTc interval or seizures, by modulating VGSCs function. In this study, we investigated the effects of escitalopram on Na_v1.2 VGSCs expressed predominantly in the central nervous system using mouse SCN2A transfected HEK293 cells.

MATERIALS AND METHODS

Generating HEK293 Cells Expressing Na_v1.2 VGSCs and Cell Culture

The cDNA of Na_v1.2 VGSCs was gifted from Dr. Masaharu Noda.¹¹⁾ In brief, the cDNA of Na_v1.2 VGSCs inserted into pCI-neo Mammalian Expression Vector (Promega, Fitchburg, WI, U.S.A.) was transfected with FuGENE^® 6 Transfection Reagent (Promega) to HEK293 cells (JCRB Cell Bank, Osaka, Japan) as following the manufacturer’s instruction. Cells were cultured as described previously with a slight modificaition.¹²⁾ To select HEK293 cells expressing Na_v1.2 VGSCs, cells were cultured with media containing 500 µg/mL Geneticin (Nacalai Tesque, Inc., Kyoto, Japan).

Electrophysiology

Whole-cell VGSCs current were recorded as described previously.¹²⁾ All experiments were performed at 22–25°C in a bath solution containing (mM) 100 NaCl, 40 tetraethylammonium (TEA)-Cl, 0.03 CaCl₂, 10 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 10 MgCl₂·6H₂O and 10 D-glucose, and the pH was adjusted to 7.4 with NaOH. The microelectrode solution consisted of (mM) 115 CsCl, 25 NaCl, 2 MgCl₂·6H₂O, 1 CaCl₂, 11 ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and 10 HEPES, and the pH was adjusted to 7.4 with CsOH. The patch microelectrodes used for recording were made from borosilicate capillary glass (Narishige, Tokyo, Japan) and had resistances of 3–5 MΩ. Whole-cell current were recorded using an EPC-7 plus amplifier (HEKA Elektronik, Lambrecht, Germany) with low-pass filtering at 3 kHz and digitized with a Digidata1440A (Molecular Devices, Sunnyvale, CA, U.S.A.). Leak current were subtracted by a P/4 pulse protocol. These recorded data were sampled and analyzed using pCLAMP10.6 software (Molecular Devices). Escitalopram oxalate was obtained from H. Lundbeck A/S (Batch: V 4013, Copenhagen, Denmark) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution. The final concentration of DMSO in the bath solution was set at 0.1%. To explore the effect of escitalopram on Na_v1.2 VGSCs current, the bath was perfused with bath solution in which escitalopram was dissolved at the indicated concentration.

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±standard deviation (S.D.) As statistical analysis, One-way ANOVA was performed followed by Dunnett multiple comparisons test to compare the effect of various concentration of escitalopram with vehicle treatment. In addition, Student’s paired t-test was performed for statistical analysis to compare various parameters of activation or inactivation curves fitted by Boltzmann function between before and after treatment of escitalopram.

RESULTS

First, we investigated the effect of escitalopram on Na_v1.2 VGSCs current. Figure 1A shows the effect of 100 µM escitalopram on the Na_v1.2 VGSCs current evoked by depolarization from a holding potential of −80 mV to a testing potential from −100 mV to 40 mV in steps of 10 mV in HEK293 cells. Under treatment with 100 µM escitalopram, the Na_v1.2 VGSCs current decreased by around 50% compared to those before treatment with escitalopram (Fig. 1A). Figure 1B shows the current–voltage relationship before and after treatment with 100 µM escitalopram and after washing out. In cells with a peak current at −10 mV, the inhibitory effect of 100 µM escitalopram started at around −20 mV and reached a maximum at −10 mV, after which inhibition persisted with further depolarization potentials (Fig. 1B). After washing out of escitalopram, the inhibitory effect of escitalopram on Na_v1.2 VGSCs current was almost fully restored (Figs. 1A, 1B). Treatment with 10, 30, 100, 300 and 500 µM escitalopram decreased Na_v1.2 VGSCs peak current by 19.2±7.6, 28.3±7.5, 50.7±8.3, 76.5±0.5 and 82.5±5.6% compared to that before treatment with escitalopram, respectively. Compared to treatment with vehicle alone, treatment with more than 30 µM of escitalopram significantly inhibited Na_v1.2 VGSCs peak current (Fig. 1C). When the inhibitory effect toward Na_v1.2 VGSCs peak current obtained under treatment with 500 µM escitalopram was taken to represent a maximal inhibitory response and those data sets were fitted with a logistic function; L/1+exp^−k(X−X0), the IC₅₀ was determined to be 114.2 µM. When lamotrigine, which is known as an antiepileptic drug and inhibitor of VGSCs including Na_v1.2, was treated in the same experimental condition, Na_v1.2 VGSCs current was also inhibited in dose-dependent manner (supplemental data). Figure 2 shows the effect of escitalopram on the activation and inactivation of Na_v1.2 VGSCs. The voltage at half-maximal activation changed significantly from −13.8±4.6 to −21.5±3.9 mV under treatment with 100 µM escitalopram. In addition, the slope factor (k) of the activation curve also changed significantly from −7.7±2.3 to −5.7±1.3 (Fig. 2A). The voltage at half-maximal inactivation also changed significantly from −50.3±3.7 to −56.7±6.0 mV under treatment with 100 µM escitalopram. In contrast, there was no significant difference in the slope factor of the inactivation curve between before (5.7±1.9) and after treatment with 100 µM escitalopram (6.1±1.4) (Fig. 2B).

Fig. 1. Effect of Escitalopram on Na_v1.2 VGSCs Current

(A) Current traces recorded from HEK293 cells expressing rat Na_v1.2 VGSCs before and after treatment with 100 µM escitalopram and after washing out of escitalopram. To record Na_v1.2 VGSCs current, cells were held at −80 mV and stepped to a test pulse (from −100 to 40 mV) for 40 ms in steps of 10 mV. (B) Current–voltage relationship of normalized Na_v1.2 VGSCs current. The normalized peak Na_v1.2 VGSCs current before treatment (open circle), under treatment with 100 µM escitalopram (filled circle) and after washing out of escitalopram (filled square) were plotted against each depolarizing potential. Each current–voltage relationship curve represents the mean±S.D. (n=9). (C) Dose-dependent effect of escitalopram on Na_v1.2 VGSCs current. Histograms show the relative Na_v1.2 VGSCs peak currents under treatment with vehicle (n=4), or 10 (n=4), 30 (n=5), 100 (n=15), 300 (n=4) and 500 µM escitalopram (n=9). Histograms represent the mean±S.D. (n=4–15, *; p<0.05 vs. vehicle).

Fig. 2. Effects of Escitalopram on the Activation and Inactivation of Na_v1.2 VGSCs

(A) Activation was determined from current–voltage relationships by normalizing peak Na_v1.2 VGSCs current to the driving force and maximal Na_v1.2 VGSCs current, and plotting normalized conductance versus the potential of the depolarizing pulse. The conductances of Na_v1.2 VGSCs were calculated as G=I (E−E_rev). These data sets, before (open circle) and after treatment with 100 µM escitalopram (filled circle), were fitted with a Boltzmann function; 1/1+exp^((V−Vh)/k). Each activation curve represents the mean±S.D. (n=15). (B) To record Na_v1.2 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 was determined by plotting the normalized peak current during the following test pulse at −10 mV for 20 ms versus the pre-pulse potential. These data sets, before (open circle) and after treatment with 100 µM escitalopram (filled circle), were fitted with a Boltzmann function. Each inactivation curve represents the mean±S.D. (n=8).

DISCUSSION

The present study was designed to update our understanding of SSRIs which might have other target molecule except for serotonin transporter. Thus, we investigated whether or not escitalopram affected Na_v1.2 VGSCs current. Although escitalopram is an SSRI and has been considered to not interact with other biological molecules including VGSCs, we demonstrated that escitalopram inhibited the Na_v1.2 VGSCs current amplitude in a dose-dependent manner. Moreover, escitalopram also caused a significant hyperpolarizing shift in both the activation and inactivation curves of Na_v1.2 VGSCs. It has been reported that citalopram, which is a racemic mixture of (R)-(−)-citalopram and (S)-(+)-citalopram, inhibited both Na_v1.7 and Na_v1.8 VGSCs current, with IC₅₀ values of 174 and 100 µM, respectively.¹³⁾ In addition, it has been shown that both citalopram and escitalopram may cause QRS prolongation via sodium channels.¹⁴⁾ These findings suggest that it is reasonable for escitalopram to have an inhibitory effect on Na_v1.2 VGSCs current. However, it is also fact that IC₅₀ of escitalopram against Na_v1.2 VGSCs was pretty high when the plasma concentration of escitalopram was considered for the clinical treatment. It has been reported that postmortem serum, which was obtained from a woman presented to the emergency department after having witnessed seizures, contained 7300 ng/mL citalopram.¹⁵⁾ Therefore, the side effects of escitalopram via the modulation of Na_v1.2 VGSCs current might be caused when escitalopram is given as an overdose or is used in patients with a genetic modification in VGSCs, such as epileptic patients.^3,7) Moreover, it has been considered that the genetic modification of Na_v1.2 related to generate the generalized epilepsy with febrile seizures.^8,16) This result also may support that the side effect induced by escitalopram, such as epilepsy, might be caused via the functional modification of Na_v1.2. In contrast, it also has been known three types of VGSCs, such as Na_v1.1, Na_v1.3 and Na_v1.6 other than Na_v1.2, that are predominantly expressed in the central nervous system. Considering the effect of antiepileptic drugs in various VGSCs expressed in central nervous system,¹⁷⁾ escitalopram also may inhibit not only Na_v1.2 but also other subtype of VGSCs in central nervous system in similar manner. In this study, it has also been demonstrated that escitalopram demonstrated the hyperpolarized shift in the voltage-dependence of both activation and inactivation. Several antiepileptic drugs or local anesthesia have demonstrated the hyperpolarized shift in the voltage-dependence of inactivation.^16,17) In this respect, the inhibition mode of escitalopram may be similar to such drugs in the inactivation. Recently, it has been reported that a certain genetic mutation of Na_v1.2 which demonstrated the hyperpolarized shift in the voltage-dependence of both activation and inactivation was identified in a patient with sporadic neonatal epileptic encephalopathy.¹⁸⁾ Considering these studies, the inhibition mode of escitalopram that affected both of activation and inactivation states on Na_v1.2 may be unique and induce a similar condition like a patient with epileptic encephalopathy mentioned above by the hyperpolarized shift in the voltage-dependence of both activation and inactivation. Though it is puzzling to explain how those phenomena affect the function of Na_v1.2, the shift of the voltage-dependence of both activation and inactivation toward negative potential may lead the imbalance of sodium influx followed by the disturbance of normal excitation of neurons at a certain range of membrane potential.

In summary, escitalopram inhibited Na_v1.2 VGSCs current in a dose-dependent manner. Moreover, both voltage-dependence of the activation and inactivation were shifted toward hyperpolarization by treatment with escitalopram. Further studies will be needed to clarify the correlation between the electrophysiological mechanisms of the inhibitory effect of escitalopram on Na_v1.2 VGSCs and its pharmacological action.

Acknowledgments

We thank the members of the Department of Pharmacotherapeutics, School of Pharmacy, International University of Health and Welfare, for their helpful discussions throughout this study. This work was supported in part by grants from the Japan Society for the Promotion of Science KAKENHI (Grant-in-Aid for Scientific Research (C), 26460703, 2014, and Grant-in-Aid for Young Scientists (B), 15K18878, 2015).

Conflict of Interest

Yoshihiko Nakatani received a research Grant from GlaxoSmithKline K.K. (GSK Japan Research Grant 2017). Taku Amano declares no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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