Epilepsy-Induced High Affinity Blockade of the Cardiac Sodium Current INa by Lamotrigine; A Potential for Acquired Arrythmias

Lamotrigine is widely prescribed to treat bipolar neurological disorder and epilepsy. It exerts its antiepileptic action by blocking voltage-gated sodium channels in neurons. Recently, the US Food and Drug Administration issued a warning on the use of Lamotrigine after observations of conduction anomalies and Brugada syndrome patterns on the electrocardiograms of epileptic patients treated with the drug. Brugada syndrome and conduction disturbance are both associated with alterations of the cardiac sodium current (INa) kinetics and amplitude. In this study, we used the patch clamp technique on cardiomyocytes from epileptic rats to test the hypothesis that Lamotrigine also blocks INa in the heart. We found that Lamotrigine inhibited 60% of INa peak amplitude and reduced cardiac excitability in epileptic rats but had little effect in sham animals. Moreover, Lamotrigine inhibited 67% of INaL and, more importantly, prolonged the action potential refractory period in epileptic animals. Our results suggest that enhanced affinity of Lamotrigine for INa may in part explain the clinical phenotypes observed in epileptic patients.


Introduction
Lamotrigine (Lamictal™, LTG) is an antiepileptic drug approved by the US Food and Drug Administration (FDA) in 1994 to treat bipolar disorders and epilepsy. Part of the antiepileptic action of LTG stems from its ability to block the sodium current [1,2] and possibly the neuronal transient outward potassium current IA [3] expressed in the cortex and hippocampus of the brain. The overall effect is to stabilize the presynaptic membrane of neuronal cells and to attenuate the effect of the excitatory neurotransmitter glutamate during repetitive neuronal firing [4]. Slow binding of LTG during activation or fast inactivation of neuronal sodium channels (nNa V s) was proposed to explain the block of sodium current in neurons [2,[5][6][7]. Whether LTG also blocks the cardiac sodium current (I Na ) at therapeutic doses (100-500 mg/day) yielding an effective plasma concentration between 2-16 µg/mL (7.8-62.5 µmol/L) [8][9][10], is unknown. However, clinical observations of Brugada syndrome (BrS) phenotype [11][12][13][14] and QRS prolongation [15] on the electrocardiograms (ECG) of epileptic patients treated with LTG are consistent with conduction disturbances and I Na block. Those observations, coupled with a series of case reports on LTG cardiotoxicity [16][17][18], prompted the FDA to restrict the use of LTG in 2021 and to add a warning label stating that "Lamictal exhibits class 1B antiarrhythmic activity at therapeutically relevant concentrations." [19]. The mechanism by which LTG may exert its arrhythmogenic effect remains unknown. Identifying such potential mechanisms linked to its effect on I Na is the goal of this paper.
In sharp contrast to epileptic patients, studies from toxicology centers indicate that non-epileptic patients on LTG overdose, with serum levels between 17-90 µg/mL, exhibit only minor to moderate neurologic or electrocardiographic effects [18]. This raises the possibility that epilepsy enhances the apparent affinity of LTG for cardiac I Na , possibly by enhancing expression of nNavs in the heart. In support, we previously reported that epilepsy increases the contribution of nNa V s to I Na [20,21]. Because of the apparent higher affinity of LTG for neuronal cells, we wanted to test if enhanced expression of nNa V s in the heart ventricle may contribute to the arrhythmogenic event clinically reported in epileptic patients.
In this study, we present a series of experiments showing that indeed, epilepsy increased the affinity of LTG for I Na . The enhanced blockade of I Na also prolonged the action potential refractory period. The results may provide a basis to explain the conduction disturbances and the BrS phenotype associated with LTG toxicity in epileptic patients.

Results
We first compared the effects of LTG on I Na in ventricular cardiomyocytes from epileptic and sham animals. Figure 1A shows that 10 µmol/L of LTG decreased I Na amplitude more for epileptic animals. Blockade of peak I Na was 16 ± 6% and 61 ± 6% for sham and epileptic animals, respectively. Current voltage relationships ( Figure 1B) showed that LTG blockade of I Na by 10 µmol/L and 100 µmol/L was similar in epileptic animals. LTG (10 µmol/L) also shifted voltage dependence of I Na activation in epileptic animals but not in sham. Mid-activation voltage (V 0.5 ) was depolarized by 5 mV (Table 1, Figure 1C) in cardiomyocytes from epileptic animals. I Na voltage threshold (I Na,Th ) was depolarized by 5 ± 1 mV and 10.7 ± 1.4 mV with 10 and 100 µmol/L of LTG, respectively, in epileptic animals ( Figure 1D), while I Na,Th was depolarized 8.5 ± 1.5 mV by 100 µmol/L of LTG in sham animals. The maximum Na + conductance (G Na,max ) decreased by 49 ± 5% in the presence of LTG (10 µmol/L) in epileptic animals but had no effect in the sham group ( Figure 1E). Table 1. Na + channel activation parameters for control conditions and during exposure to 10 µmol/L of LTG. Tukey test using one-way ANOVA (ctrl vs. LTG), * indicates p < 0.05.

Lamotrigine Blockade of I Na
Our results from Figure 1, showing that blockade of I Na by LTG was larger in epileptic animals, combined with our previous data indicating an increased contribution of TTXsensitive Na + channels [20] during epilepsy, suggest that LTG may target at least two populations of Na + channels in ventricular cardiomyocytes. Accordingly, this should translate into a biphasic dose-response curve for I Na . Figure 2A shows that blockade of I Na by LTG in sham animals could be reasonably fit to a single monophasic Hill equation, thus, suggesting that blockade of I Na follows mostly single receptor binding kinetics. In sharp contrast, epileptic rats exhibited a biphasic response to LTG with a high affinity blockade generating a plateau at 47% of the maximum amplitude for concentrations between 10-100 µmol/L and a sensitivity like that of sham animals for larger concentrations. These results indicate at least two LTG binding sites contributed to block I Na during epilepsy. LTG half-maximal blocking concentration (IC 50 ) obtained from the fit to data was 155 ± 22 µmol/L in sham animals. In epileptic conditions, IC 50 values were 211 ± 48 µmol/L and 1.5 ± 0.3 µmol/L Pharmaceuticals 2022, 15, 1208 3 of 14 ( Figure 2B). This result, therefore, indicates an increased contribution of LTG-sensitive channels to I Na .
We first compared the effects of LTG on INa in ventricular cardiomyocytes from epileptic and sham animals. Figure 1A shows that 10 µ mol/L of LTG decreased INa amplitude more for epileptic animals. Blockade of peak INa was 16 ± 6% and 61 ± 6% for sham and epileptic animals, respectively. Current voltage relationships ( Figure 1B) showed that LTG blockade of INa by 10 µ mol/L and 100 µ mol/L was similar in epileptic animals. LTG (10 µ mol/L) also shifted voltage dependence of INa activation in epileptic animals but not in sham. Mid-activation voltage (V0.5) was depolarized by 5 mV (Table 1, Figure 1C) in cardiomyocytes from epileptic animals. INa voltage threshold (INa,Th) was depolarized by 5 ± 1 mV and 10.7 ± 1.4 mV with 10 and 100 µ mol/L of LTG, respectively, in epileptic animals ( Figure 1D), while INa,Th was depolarized 8.5 ± 1.5 mV by 100 µ mol/L of LTG in sham animals. The maximum Na + conductance (GNa,max) decreased by 49 ± 5% in the presence of LTG (10 µ mol/L) in epileptic animals but had no effect in the sham group ( Figure 1E).

Lamotrigine Prolonged Refractory Period
We next tested if the reduction in I Na caused by LTG could be due to a decrease in Na + channel availability (steady-state inactivation). At therapeutic concentration (10 µmol/L), LTG hyperpolarized mid-inactivation potential (V h ) of I Na by 2.7 mV and 6.7 mV in sham and epileptic animals, respectively ( Figure 3, Table 2). Those shifts in steady-state inactivation would theoretically reduce I Na amplitude by 12% and 18% in sham and epileptic animals when cardiomyocytes are sitting around their natural resting membrane potential of −80 mV and may contribute to a reduction in cell excitability.
trast, epileptic rats exhibited a biphasic response to LTG with a high affinity blockade generating a plateau at 47% of the maximum amplitude for concentrations between 10-100 µ mol/L and a sensitivity like that of sham animals for larger concentrations. These results indicate at least two LTG binding sites contributed to block INa during epilepsy. LTG half-maximal blocking concentration (IC50) obtained from the fit to data was 155 ± 22 µ mol/L in sham animals. In epileptic conditions, IC50 values were 211 ± 48 µ mol/L and 1.5 ± 0.3 µ mol/L ( Figure 2B). This result, therefore, indicates an increased contribution of LTGsensitive channels to INa.

Lamotrigine Prolonged Refractory Period
We next tested if the reduction in INa caused by LTG could be due to a decrease in Na + channel availability (steady-state inactivation). At therapeutic concentration (10 µ mol/L), LTG hyperpolarized mid-inactivation potential (Vh) of INa by 2.7 mV and 6.7 mV in sham and epileptic animals, respectively ( Figure 3, Table 2). Those shifts in steady-state inactivation would theoretically reduce INa amplitude by 12% and 18% in sham and epileptic animals when cardiomyocytes are sitting around their natural resting membrane potential of −80 mV and may contribute to a reduction in cell excitability.   Table 2. Na + channel inactivation parameters for control conditions and during exposure to 10 µmol/L of LTG. Tukey test using one-way ANOVA (ctrl vs. LTG), * indicates p < 0.05, ** p < 0.01.

Sham V h (mV) Slope
I Na recovery from inactivation plays an important role in regulating the minimal time needed between successive action potentials (refractory period). Using standard recovery protocols ( Figure 4A), we found that I Na recovery was best described by a sum of two exponentials. Fitting data to a two-exponential function showed that epilepsy alone delayed recovery by increasing the slow recovery time constant by 20%. LTG only slightly slowed recovery of I Na in sham and epilepsy cardiomyocytes ( Figure 4B, Table 3).
INa recovery from inactivation plays an important role in regulating the minimal time needed between successive action potentials (refractory period). Using standard recovery protocols ( Figure 4A), we found that INa recovery was best described by a sum of two exponentials. Fitting data to a two-exponential function showed that epilepsy alone delayed recovery by increasing the slow recovery time constant by 20%. LTG only slightly slowed recovery of INa in sham and epilepsy cardiomyocytes ( Figure 4B, Table 3).  The refractory period is caused by the time needed for the channels to transit back from the inactivated to the closed state, from which they can re-open. We next tested  The refractory period is caused by the time needed for the channels to transit back from the inactivated to the closed state, from which they can re-open. We next tested whether those effects of LTG on I Na recovery translated into a longer refractory period of cardiomyocyte action potentials ( Figure 5A,C). In control conditions, the time needed to recover a full amplitude action potential at resting membrane voltages of −80 mV or −100 mV was similar between sham and epileptic animals ( Figure 5B,D). LTG (1 µmol/L) increased the total refractory period by 65 ± 11% and 185 ± 13% at −80 mV, and by Pharmaceuticals 2022, 15, 1208 6 of 14 106 ± 17% and 173 ± 26% at −100 mV in sham and epileptic animals, respectively. These results indicate that LTG increased the refractory period by a factor of 1.9 for sham and 2.8 for epileptic animals (p < 0.001, Tukey test with one-way ANOVA). As previously reported, epileptic rats displayed longer action potentials [20] (Figure S1) at the plateau level in part due to a larger window current ( Figure S2). LTG (1 µmol/L) reduced early action potential duration (30% repolarization) by 30.4 ± 4.2% and 33.5 ± 4.2% in sham and epileptic animals, respectively. We next evaluated if the effects of LTG on I Na late current could account for the changes in APD. Our measurements using a voltage ramp protocol showed that epilepsy increased I NaL by 26 ± 9.2%, from −1.5 ± 0.2 to −1.9 ± 0.1 pA/pF ( Figure 6A), as we previously reported [20,21]. Following application of 10µmol/L of LTG, I NaL amplitude was reduced from −1.5 ± 0.2 to −1.4 ± 0.2 pA/pF and from −1.9 ± 0.1 to −0.6 ± 0.1 pA/pF in sham and epileptic animals, respectively ( Figure 6B). This reduction in I NaL represents a block of 6.7 ± 4.3% in sham and 66.7 ± 5.8% during epilepsy. LTG at 10 µmol/L decreased the window current calculated from the overlap of the activation and steady-state inactivation of I Na by 9.5% and 91.3% in sham and epileptic animals, respectively ( Figure S2). These values are close to those found experimentally with our I NaL measurements. Overall, our data show a strong contribution of I NaL blockade to modulation of APD by LTG. whether those effects of LTG on INa recovery translated into a longer refractory period of cardiomyocyte action potentials ( Figure 5A,C). In control conditions, the time needed to recover a full amplitude action potential at resting membrane voltages of −80 mV or −100 mV was similar between sham and epileptic animals ( Figure 5B,D). LTG (1 µ mol/L) increased the total refractory period by 65 ± 11% and 185 ± 13% at −80 mV, and by 106 ± 17% and 173 ± 26% at −100 mV in sham and epileptic animals, respectively. These results indicate that LTG increased the refractory period by a factor of 1.9 for sham and 2.8 for epileptic animals (p < 0.001, Tukey test with one-way ANOVA). As previously reported, epileptic rats displayed longer action potentials [20] ( Figure S1) at the plateau level in part due to a larger window current ( Figure S2). LTG (1 µ mol/L) reduced early action potential duration (30% repolarization) by 30.4 ± 4.2% and 33.5 ± 4.2% in sham and epileptic animals, respectively. We next evaluated if the effects of LTG on INa late current could account for the changes in APD. Our measurements using a voltage ramp protocol showed that epilepsy increased INaL by 26 ± 9.2%, from −1.5 ± 0.2 to −1.9 ± 0.1 pA/pF ( Figure 6A), as we previously reported [20,21]. Following application of 10µ mol/L of LTG, INaL amplitude was reduced from −1.5 ± 0.2 to −1.4 ± 0.2 pA/pF and from −1.9 ± 0.1 to −0.6 ± 0.1 pA/pF in sham and epileptic animals, respectively ( Figure 6B). This reduction in INaL represents a block of 6.7 ± 4.3% in sham and 66.7 ± 5.8% during epilepsy. LTG at 10µ mol/L decreased the window current calculated from the overlap of the activation and steady-state inactivation of INa by 9.5% and 91.3% in sham and epileptic animals, respectively ( Figure S2). These values are close to those found experimentally with our INaL measurements. Overall, our data show a strong contribution of INaL blockade to modulation of APD by LTG.
In the heart, sodium channels contribute to conduction and cellular excitability by modulating the voltage threshold, the rising phase, and the duration of action potentials. Although the bulk of sodium current is carried by the cardiac isoform NaV1.5, we previously showed that tetrodotoxin TTX-sensitive channels contribute to INa and INaL [21,26,27] This contribution increased during epilepsy. We previously reported contributions of ≈ 19% and ≈ 35% to INaL for epileptic rats. Our data (Figure 1) show that blockade of INa is more important in cardiomyocytes from epileptic animals. Therefore, our observation of a saturating block by LTG is most likely linked to blockade of neuronal channel isoforms overexpressed during epilepsy and contributing to INa. Those data are consistent with our previous RT-PCR and Western blot analyses showing that cDNA and protein expression of neuronal sodium channels, especially NaV1.1, is enhanced in cardiac ventricles of epileptic animals [20].

Discussion
The Na + channel family consists of 10 pore-forming α-subunits associated with auxiliary β-subunits identified as β 1 through β 4 [22]. Among them, tetrodotoxin (TTX)-sensitive voltage-gated channels Na V 1.1, Na V 1.2, Na V 1.3 and Na V 1.6 are the most abundantly expressed in the brain, while the TTX-resistant Na V 1.5 is considered the cardiac isoform [23]. Overexpression of Na V 1.1 and Na V 1.3 within the brain has been reported in non-hereditary forms of epilepsy [24,25]. In a previous study, we demonstrated that epilepsy also enhances expression of TTX-sensitive channels (among them, Na V 1.1) within the rat cardiac ventricle.
In the heart, sodium channels contribute to conduction and cellular excitability by modulating the voltage threshold, the rising phase, and the duration of action potentials. Although the bulk of sodium current is carried by the cardiac isoform Na V 1.5, we previously showed that tetrodotoxin TTX-sensitive channels contribute to I Na and I NaL [21,26,27] This contribution increased during epilepsy. We previously reported contributions of ≈19% and ≈35% to I NaL for epileptic rats. Our data (Figure 1) show that blockade of I Na is more important in cardiomyocytes from epileptic animals. Therefore, our observation of a saturating block by LTG is most likely linked to blockade of neuronal channel isoforms overexpressed during epilepsy and contributing to I Na . Those data are consistent with our previous RT-PCR and Western blot analyses showing that cDNA and protein expression of neuronal sodium channels, especially Na V 1.1, is enhanced in cardiac ventricles of epileptic animals [20].
However, we were surprised by the amplitude of the block by LTG during epilepsy. Based on our previous results showing that 35% of peak I Na is generated by TTX-sensitive Pharmaceuticals 2022, 15, 1208 8 of 14 channels in epileptic animals, we were expecting a proportional level of I Na block by LTG. The 61% block shown on the peak I Na -I/V curve and the plateau at 53% block on the dose-response curve for epileptic animals (Figure 2) indicates that some Na V 1.5 channels might also have been blocked but with a lower affinity, or part of the blockade is due to the inactivation kinetics of I Na .
The modulated receptor hypothesis of Hille [28] may explain blockade of neuronal sodium channels by LTG. Under this model, LTG binds to open channels and stabilizes inactivation in a higher affinity block [6,[29][30][31][32]. This mechanism is generally thought to be responsible for the use-dependent block of I Na by local anesthetics such as lidocaine [33]. It was proposed to explain the antiepileptic effect of LTG by accumulation of I Na block in the inactivated states during rapid firing in hippocampus [1]. Such a mechanism is consistent with our data showing minimal alterations of steady-state activation in both sham and epileptic animals but a significant hyperpolarization of I Na mid-inactivation potential during epilepsy (Figure 3), as previously reported [2]. The higher affinity block in the inactivated state may also explain the slower recovery of I Na in both sham and epileptic animals. Given that our pulse protocol to measure I Na was 25 ms, nNavs had more time to inactivate, and this may have increased blockade by LTG at lower concentrations. Therefore, the saturating effect of LTG block can be explained by a low-affinity blockade of cardiac Na V 1.5 channels and a high-affinity blockade of nNavs overexpressed during epilepsy.
A key difference between neuronal and cardiac action potentials is their duration (APD). In neurons from the central nervous system, APD is between 1 and 10 ms [34] as opposed to cardiac ventricular APD, which will vary from 20-80 ms in rodents and up to 450 ms in humans [35]. Therefore, any neuronal sodium channel isoform sensitive to LTG will have a much longer inactivation time in the heart, and this will promote a more important block of I Na . Potentiation of I Na blockade may significantly reduce conduction and potentiate ventricular bradycardia by prolonging the refractory period and reducing APD, as observed during our action potential measurements in cardiomyocytes from epileptic animals.
Interestingly, the reduction in APD occurs at 30% and 50% repolarization ( Figure S1), which is the region of major I NaL involvement and is more important in epileptic animal vs. sham. In agreement, we found a larger reduction in I NaL during epilepsy. Prolongation of the refractory period is anti-arrhythmic in cases where heart rate is rapid, such as in tachycardia and polymorphic VT. In those type of arrythmias, the beneficial effect comes from slowing the accelerated heart rate or reducing dispersion of electrical repolarization within the ventricle. However, in normal settings, an increase in I Na refractory period could prevent subsequent beats from occurring normally and slow cardiac rhythm to rates that cause bradycardia. In those conditions, slowing recovery of I Na may, therefore, potentiate cardiac arrhythmias. This represents a potential mechanism that may explain the clinical observations of bradycardia and conduction problems in patients treated with LTG.
Late sodium current contributes to AP repolarization plateau, and its enhancement causes a longer QT interval, which triggers arrhythmias. Our results show that epilepsy increased I NaL amplitude by 27%, a value close to what we already reported [20]. Interestingly, LTG at therapeutic concentration (10 µmol/L) reduced I NaL by 66.7 ± 5.8% only in epileptic animals (Figure 6), further suggesting a significant contribution of LTG-sensitive nNavs during epilepsy. Our results show that epilepsy increased blockade of I NaL by LTG by a factor of 10. I NaL inhibition could have benefits against pathologies such as druginduced or hereditary long QT syndrome [35] by reducing the duration of the ventricular AP duration. We previously reported that epilepsy prolonged ventricular APD by increasing I NaL . Our results suggest that LTG may prevent or reduce QT prolongation in epileptic individuals. However, this protective effect may be lost when combined with blockade of the I Na peak (61%). Indeed, a reduction in peak I Na will impact the early phase 1 of the AP by disturbing the balance between I Na and the transient outward potassium current (I to ), especially in the epicardial layer of the ventricle. The reduction in I Na amplitude will potentiate early repolarization by I to . Adding the APD shortening effect of weaker I NaL is likely to further promote early repolarization, a hallmark of BrS. In regions, such as the epicardium of the right ventricle, where I to is most prominent, repolarization will occur rapidly, while the inner part of the ventricle will remain fully depolarized [36]. This dispersion of repolarization within the cardiac ventricle can trigger shunt currents from the still depolarized areas to the fully repolarized regions. Consequently, the fully repolarized regions of the epicardium may re-excite prematurely, thus creating a substrate for re-entrant arrhythmias. This may in part explain the observations of BrS arrhythmias in epileptic patients treated with LTG.
Overall, our data demonstrate that LTG, by virtue of its effect on I Na , may cause a reduction in cardiac conduction speed, excitability and APD. Those effects are consistent with clinical observations of QRS widening [18,[37][38][39] during LTG overdose and BrS phenotypes [11,12,36,40,41] in epileptic patients. Moreover, because of the enhanced expression of nNavs sensitive to LTG during epilepsy, our data may in part explain why cardiac disturbances seem relatively benign during overdose in patients treated for depression-related illness but may become serious and potentially deadly for epileptic patients. Our study thus provides a basic framework to explain the increased risk of cardiac arrhythmias seen in epileptic patients under treatment with LTG.

Animal Model and Cell Dissociation
Epilepsy was induced in rats by subcutaneous injection of Kainic acid (KA, Hello Bio Inc., Princeton, NJ, USA) as previously described [20]. Briefly, adult Sprague Dawley rats (~250-275 g) were injected twice with 8 mg/kg of KA, with a 1.5 to 2 h interval between injections, to induce seizures. The status epilepticus was stopped by intraperitoneal (IP) injection of 25 mg/kg of diazepam (Valium, Rexall Pharmacy Group Ltd., Toronto, ON, Canada) 2 h after KA. Animals were constantly monitored for 36-40 days after induction of epilepsy. Only animals showing chronic seizure behavior consisting of rearing and falling corresponding to stages 4 and 5 on the Racine scale over the course of 36 day were used in this study [42]. Sham animals received diazepam and saline in lieu of KA. Animals were housed 1 per cage on a 14 h/10 h light/dark cycle with free access to tap water and food. All animals were used between 36 and 40 days after treatment. Left ventricular cardiomyocytes from adult rats were isolated by enzymatic dissociation as we previously described [26,43]. Animals were euthanized by exsanguination. Briefly, rats were injected intraperitoneally with 500 U/kg of Sodium Heparin (Sandoz Canada Inc., Boucherville, QC, Canada) 20 min prior to sedation with 1-chloro-2,2,2-trifluoroethyl difluoro methyl ether (Isoflurane, USP, Baxter, Simcoe County, ON, Canada). When the animal was fully anesthetized, the chest was opened, and the heart rapidly excised.

Electrophysiology
I Na was recorded from ventricular cardiomyocytes at room temperature with the patch clamp technique in whole cell configuration using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Analysis was performed using the Clampex 10.7 analysis software. Recordings were acquired at 10 kHz and filtered to 5 kHz (lowpass Bessel filter). Whole cell capacitance and series resistance compensation (85%) were optimized to reduce the capacitive artifact and minimize voltage clamp error.
Cells from both sham and epileptic animals were incubated for 10 min with various concentrations of LTG and perfused constantly during patch clamp measurements. I Na recordings took around 20 min. The quality of the giga-seal after the 20 min was, in most cases, not good enough to perform LTG washout and attempt to record the current again.
For action potential (AP) measurements, membrane potential was kept at either −100 mV or −80 mV in current clamp mode (I = 0). Action potentials were triggered by 1 ms pulses of threshold current applied at a frequency of 20 Hz where mentioned. It is important to note that in the LTG experiments, it was necessary to perform the AP measurements with 1 µmol/L instead of 10 µmol/L, because the AP was not triggered in the presence of 10 µmol/L, which introduced substantial variability in the results.

Materials
Lamotrigine (LTG, ApexBio LLC Technology, Houston, TX, USA) was dissolved in DMSO to make a stock solution of 0.3 mol/L, from which we diluted in extracellular solution to reach the final concentrations. DMSO in LTG extracellular solutions never exceeded 0.2%. No DMSO effects on I Na were observed at this maximum concentration.

Data Analysis
Figures were produced and data analysis performed with the pCLAMP program suite software (Molecular Devices, Sunnyvale, CA, USA) and Origin 8.5 (OriginLab, Nothampton, MA, USA). I Na current voltage relationships were obtained from recordings during 25 ms voltage steps between −70 and 10 mV, in 5 mV increments, from a −100 mV holding potential. Average peak current values were normalized to membrane capacitance (Cm) and plotted against the test voltage to construct the I Na current-voltage curves (pA/pF). Conductance (G Na ) was calculated as G Na = I Na /(Vm − E Na ), where Vm is the membrane potential (mV) and E Na is the sodium reversal potential (mV). Maximum conductance (G Na,Max ) was obtained from the slope of a linear regression fit to the linear portion of the I/V at voltage more positive than −25 mV.
Steady-state inactivation was measured using a 15 ms test pulse to 30mV following a series of 500 ms conditioning potentials from −120 to −40 mV in 5 mV increments. Inactivation curves were obtained by plotting the ratio of I Na to its maximum value (I Na,Max ) as a function of the conditioning voltage.
Activation and inactivation data were fitted to a standard Boltzmann distribution function: Y = (A 1 −A 2 ) 1+[(Vm−E Na )/V 0.5 ] + A 2 , where Y represents the fraction of activated (m) or available (h) channels obtained, respectively, from the ratio of the macroscopic conductance (G Na /G Na,Max ) or the sodium current I/I Max . Vm and E Na were defined as before, and V 0.5 was the mid-potential for activation or inactivation. G Na was obtained from the currentvoltage relationship as G Na = I Na /(Vm − E Na ), and G Na,Max represented the maximal Na + conductance. V 0.5 was the membrane voltage, where the distribution was half-maximal, corresponding to mid-activation or mid-inactivation (V h ) voltages.
Recovery from inactivation was measured using a standard double-pulse protocol while holding cells to membrane potentials of −100 mV. I Na was elicited by two-step pulses