Abstract
Eslicarbazepine acetate (ESL) is a dibenzazepine anticonvulsant approved as adjunctive treatment for partial-onset epileptic seizures. Following first pass hydrolysis of ESL, S-licarbazepine (S-Lic) represents around 95 % of circulating active metabolites. S-Lic is the main enantiomer responsible for anticonvulsant activity and this is proposed to be through the blockade of voltage-gated Na+ channels (VGSCs). ESL and S-Lic both have a voltage-dependent inhibitory effect on the Na+ current in N1E-115 neuroblastoma cells expressing neuronal VGSC subtypes including Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7. ESL has not been associated with cardiotoxicity in healthy volunteers, although a prolongation of the electrocardiographic PR interval has been observed, suggesting that ESL may also inhibit cardiac Nav1.5 isoform. However, this has not previously been studied. Here, we investigated the electrophysiological effects of ESL and S-Lic on Nav1.5 using whole-cell patch clamp recording. We interrogated two model systems: (1) MDA-MB-231 metastatic breast carcinoma cells, which endogenously express the ‘neonatal’ Nav1.5 splice variant, and (2) HEK-293 cells stably over-expressing the ‘adult’ Nav1.5 splice variant. We show that both ESL and S-Lic inhibit transient and persistent Na+ current, hyperpolarise the voltage-dependence of fast inactivation, and slow the recovery from channel inactivation. These findings highlight, for the first time, the potent inhibitory effects of ESL and S-Lic on the Nav1.5 isoform, suggesting a possible explanation for the prolonged PR interval observed in patients on ESL treatment. Given that numerous cancer cells have also been shown to express Nav1.5, and that VGSCs potentiate invasion and metastasis, this study also paves the way for future investigations into ESL and S-Lic as potential invasion inhibitors.
1 Introduction
Eslicarbazepine acetate (ESL) is a member of the dibenzazepine anticonvulsant family of compounds which also includes oxcarbazepine and carbamazepine (1). ESL has been approved by the European Medicines Agency and the United States Federal Drug Administration as an adjunctive treatment for partial-onset epileptic seizures (2). ESL is administered orally and rapidly undergoes first pass hydrolysis to two stereoisomeric metabolites, R-licarbazepine and S-licarbazeine (S-Lic; also known as eslicarbazepine; Figure 1A, B) (3–5). S-Lic represents around 95 % of circulating active metabolites following first pass hydrolysis of ESL and is the enantiomer responsible for anticonvulsant activity (6, 7). S-Lic also has improved blood brain barrier penetration compared to R-licarbazepine (8). Although S-Lic has been shown to inhibit T type Ca2+ channels (9), its main activity is likely through blockade of voltage-gated Na+ channels (VGSCs) (10). ESL offers several clinical advantages over other older VGSC-inhibiting antiepileptic drugs, e.g. carbamazepine, phenytoin; it has a favourable safety profile (10, 11), reduced induction of hepatic cytochrome P450 enzymes (12), low potential for drug-drug interactions (13, 14), and takes less time to reach a steady state plasma concentration (15).
VGSCs are composed of a pore-forming α subunit in association with one or more auxiliary βsubunits, the latter modulating channel gating and kinetics in addition to functioning as cell adhesion molecules (16). There are nine α subunits (Nav1.1-Nav1.9), and four β subunits (β1-4) (17, 18). In postnatal and adult CNS neurons, the predominant α subunits are the tetrodotoxin-sensitive Nav1.1, Nav1.2 and Nav1.6 isoforms (19) and it is therefore on these that the VGSC-inhibiting activity of ESL and S-Lic has been described. In the murine neuroblastoma N1E-115 cell line, which expresses Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7, ESL and S-Lic both have a voltage-dependent inhibitory effect on the Na+ current (10, 20). In this cell model, S-Lic has no effect on the voltage-dependence of fast inactivation, but significantly hyperpolarises the voltage-dependence of slow inactivation (10). S-Lic also has a lower affinity for VGSCs in the resting state than carbamazepine or oxcarbazepine, thus potentially improving its therapeutic window over first- and second-generation dibenzazepine compounds (10). In acutely isolated murine hippocampal CA1 neurons, which express Nav1.1, Nav1.2 and Nav1.6 (21–23), S-Lic significantly reduces the persistent Na+ current, a very slow inactivating component ~1 % the size of the peak transient Na+ current (24, 25). Moreover, in contrast to carbamazepine, this effect is maintained in the absence of β1 (24, 26).
In healthy volunteers, ESL has not been associated with cardiotoxicity and the QT interval remains unchanged on treatment (27). However, a prolongation of the PR interval has been observed (27), suggesting that caution should be exercised in patients with cardiac conduction abnormalities (13). Prolongation of the PR interval suggests that ESL may also inhibit the cardiac Nav1.5 isoform, although this has not previously been studied. Nav1.5 is not only responsible for the initial depolarisation of the cardiac action potential (28), but is also expressed in breast and colon carcinoma cells, where the persistent Na+ current promotes invasion and metastasis (29–32). Inhibition of Nav1.5 with phenytoin or ranolazine decreases tumour growth, invasion and metastasis (33–35). Thus, it is of interest to specifically understand the effect of ESL on the Nav1.5 isoform.
In the present study we investigated the electrophysiological effects of ESL and S-Lic on Nav1.5 [1] endogenously expressed in the MDA-MB-231 metastatic breast carcinoma cell line, and [2] stably over-expressed in HEK-293 cells. We show that both ESL and S-Lic inhibit transient and persistent Na+ current, hyperpolarise the voltage-dependence of fast inactivation, and slow the recovery from channel inactivation. These findings highlight, for the first time, the potent inhibitory effects of ESL and S-Lic on the Nav1.5 isoform.
2 Materials and methods
2.1 Pharmacology
ESL (Tokyo Chemical Industry UK Ltd) was dissolved in DMSO to make a stock concentration of 67 mM. S-Lic (Tocris) was dissolved in DMSO to make a stock concentration of 300 mM. Both drugs were diluted to a working concentration of 300 μM in extracellular recording solution. The concentration of DMSO in the recording solution was 0.45 % for ESL and 0.1 % for S-Lic. Equal concentrations of DMSO were used in the control solutions. DMSO (0.45 %) had no effect on the Na+ current (Supplementary Figure 1).
2.2 Cell culture
MDA-MB-231 cells and HEK-293 cells stably expressing Nav1.5 (a gift from L. Isom, University of Michigan) were grown in Dulbecco’s modified eagle medium supplemented with 5 % FBS and 4 mM L-glutamine (36). Molecular identity of the MDA-MB-231 cells was confirmed by short tandem repeat analysis (37). Cells were confirmed as mycoplasma-free using the DAPI method (38). Cells were seeded onto glass coverslips 48 h before electrophysiological recording.
2.3 Electrophysiology
Plasma membrane Na+ currents were recorded using the whole-cell patch clamp technique, using methods described previously (32, 35). Patch pipettes made of borosilicate glass were pulled using a P-97 pipette puller (Sutter Instrument) and fire-polished to a resistance of 3-5 MΩ when filled with intracellular recording solution. The extracellular recording solution for MDA-MB-231 cells contained (in mM): 144 NaCl, 5.4 KCl, 1 MgCl2, 2.5 CaCl2, 5.6 D-glucose and 5 HEPES (adjusted to pH 7.2 with NaOH). For the extracellular recording solution for HEK-293 cells expressing Nav1.5, the extracellular [Na+] was reduced to account for the much larger Na+ currents and contained (in mM): 60 NaCl, 84 Choline Cl, 5.4 KCl, 1 MgCl2, 2.5 CaCl2, 5.6 D-glucose and 5 HEPES (adjusted to pH 7.2 with NaOH). The intracellular recording solution contained (in mM): 5 NaCl, 145 CsCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 11 EGTA, (adjusted to pH 7.4 with CsOH) (39). Voltage clamp recordings were made at room temperature using a Multiclamp 700B amplifier (Molecular Devices) compensating for series resistance by 40–60%. Currents were digitized using a Digidata 1440A interface (Molecular Devices), low pass filtered at 10 kHz, sampled at 50 kHz and analysed using pCLAMP 10.7 software (Molecular Devices). Leak current was subtracted using a P/6 protocol (40). Extracellular recording solution ± drugs was applied to the recording bath at a rate of ~1.5 ml/min using a ValveLink 4-channel gravity perfusion controller (AutoMate Scientific). Each new solution was allowed to equilibrate in the bath for ~4 min following switching prior to recording at steady state.
2.4 Voltage clamp protocols
Cells were clamped at a holding potential of −120 mV or −80 mV for ≥ 250 ms, dependent on experiment (detailed in the Figure legends). Five main voltage clamp protocols were used, as follows:
To assess the effect of drug perfusion and wash-out on peak current in real time, a simple one-step protocol was used where cells were held at −120 mV or −80 mV for 250 ms and then depolarised to −10 mV for 50 ms.
To assess the voltage-dependence of activation, cells were held at −120 mV for 250 ms and then depolarised to test potentials in 10 mV steps between −120 mV and +30 mV for 50 ms. The voltage of activation was taken as the most negative voltage which induced a visible transient inward current.
To assess the voltage-dependence of steady-state inactivation, cells were held at −120 mV for 250 ms followed by prepulses for 250 ms in 10 mV steps between −120 mV and +30 mV and a test pulse to −10 mV for 50 ms.
To assess use-dependent block, cells were held at −120 mV for 250 ms and then depolarised to 0 mV at a frequency of 50 Hz, with each depolarisation pulse lasting 5 ms.
To assess recovery from fast inactivation, cells were held at −120 mV for 250 ms, and then depolarised twice to 0 mV for 25 ms, returning to −120 mV for the following intervals between depolarisations (in ms): 1, 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 70, 100, 150, 200, 250, 350, 500. In each case, the second current was normalised to the initial current and plotted against the interval time.
2.5 Curve fitting and data analysis
To study the voltage-dependence of activation, current-voltage (I-V) relationships were converted to conductance using the following equation:
G = I / (Vm – Vrev), where G is conductance, I is current, Vm is the membrane voltage and Vrev is the reversal potential for Na+ derived from the Nernst equation. Given the different recording solutions used, Vrev for Na+ was +85 mV for MDA-MB-231 cells and +63 mV for HEK-Nav1.5 cells.
The voltage-dependence of conductance and availability were normalised and fitted to a Boltzmann equation:
G = Gmax / (1 + exp ((V1/2 – Vm) / k)), where Gmax is the maximum conductance, V1/2 is the voltage at which the channels are half activated/inactivated, Vm is the membrane voltage and k is the slope factor.
Recovery from inactivation data (It / It=0) were normalised, plotted against recovery time (Δt) and fitted to a single exponential function:
τ = A1 + A2 exp (−t / t0), where A1 and A2 are the coefficients of decay of the time constant (τ), t is time and t0 is a time constant describing the time dependence of τ.
The time course of inactivation was fitted to a double exponential function:
I = Af exp (−t / τf) + As exp (−t / τs) + C, where Af and As are maximal amplitudes of the slow and fast components of the current, τf and τs are the fast and slow decay time constants and C is the asymptote.
2.6 Statistical analysis
Data are presented as mean and SEM unless stated otherwise. Statistical analysis was performed on the raw data using GraphPad Prism 8.4.0. Pairwise statistical significance was determined with Student’s paired t-tests. Multiple comparisons were made using ANOVA and Tukey post-hoc tests, unless stated otherwise. Results were considered significant at P < 0.05.
3 Results
3.1 Effect of eslicarbazepine acetate and S-licarbazepine on transient and persistent Na+ current
Several studies have clearly established the inhibition of neuronal VGSCs (Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nav1.7 and Nav1.8) by ESL and its active metabolite S-Lic (10, 20, 24, 41). Given that ESL prolongs the PR interval (27), potentially via inhibiting the cardiac Nav1.5 isoform, together with the interest in inhibiting Nav1.5 in carcinoma cells to reduce invasion and metastasis (33, 34, 42–44), it is also relevant to evaluate the electrophysiological effects of ESL and S-Lic on this isoform. We therefore evaluated the effect of both compounds on Nav1.5 current properties using whole-cell patch clamp recording, employing a two-pronged approach: (1) recording Nav1.5 currents endogenously expressed in the MDA-MB-231 breast cancer cell line (29, 30, 45), and (2) recording from Nav1.5 stably over-expressed in HEK-293 cells (HEK-Nav1.5) (46).
Initially, we evaluated the effect of both compounds on the size of the peak Na+ current in MDA-MB-231 cells. Na+ currents were elicited by depolarising the membrane potential (Vm) to −10 mV from a holding potential (Vh) of −120 mV or −80 mV. Application of the prodrug ESL (300 μM) reversibly inhibited the transient Na+ current by 49.6 ± 3.2 % when the Vh was −120 mV (P < 0.001; n = 13; ANOVA + Tukey test; Figure 2A, D). When Vh was set to −80 mV, ESL reversibly inhibited the transient Na+ current by 79.5 ± 4.5 % (P < 0.001; n = 12; ANOVA + Tukey test; Figure 2C, E). We next assessed the effect of ESL in HEK-Nav1.5 cells. Application of ESL inhibited Nav1.5 current by 74.7 ± 4.3 % when Vh was −120 mV (P < 0.001; n = 12; Figure 2F, I) and by 90.5 ± 2.8 % when Vh was −80 mV (P < 0.001; n = 14; Figure 2H, J). However, the inhibition was only partially reversible (P < 0.001; n = 14; Figure 2F, H-J). Together, these data suggest that ESL preferentially inhibited Nav1.5 in the open or inactivated state, since the current inhibition was greater at more depolarised Vh.
We next tested the effect of the active metabolite S-Lic. S-Lic (300 μM) inhibited the transient Na+ current in MDA-MB-231 cells by 44.4 ± 6.1 % when the Vh was −120 mV (P < 0.001; n = 9; ANOVA + Tukey test; Figure 3A, D). When Vh was set to −80 mV, S-Lic (300 μM) inhibited the transient Na+ current by 73.6 ± 4.1 % (P < 0.001; n = 10; ANOVA + Tukey test; Figure 3C, E). However, the inhibition caused by S-Lic was only partially reversible (P < 0.05; n = 10; ANOVA + Tukey test; Figure 3A, C-E). In HEK-Nav1.5 cells, S-Lic inhibited Nav1.5 current by 46.4 ± 3.9 % when Vh was −120 mV (P < 0.001; n = 13; ANOVA + Tukey test; Figure 3F, I) and by 74.0 ± 4.2 % when Vh was −80 mV (P < 0.001; n = 12; ANOVA + Tukey test; Figure 3H, J). Furthermore, the inhibition in HEK-Nav1.5 cells was not reversible over the duration of the experiment. Together, these data show that channel inhibition by S-Lic was also more effective at more depolarised Vh. However, unlike ESL, channel blockade by S-Lic persisted after washout, suggesting higher target binding affinity for the active metabolite.
We also assessed the effect of both compounds on the persistent Na+ current measured 20-25 ms after depolarisation to −10 mV from −120 mV. In MDA-MB-231 cells, ESL inhibited the persistent Na+ current by 77 ± 34 % although the reduction was not statistically significant (P = 0.13; n = 12; paired t test; Figure 2B, Table 1). In HEK-Nav1.5 cells, ESL inhibited persistent current by 76 ± 10 % (P < 0.01; n = 12; paired t test; Figure 2G, Table 1). S-Lic inhibited the persistent Na+ current in MDA-MB-231 cells by 66 ± 16 % (P < 0.05; n = 9; paired t test; Figure 3B, Table 2). In HEK-Nav1.5 cells, S-Lic inhibited persistent current by 35 ± 16 % (P < 0.05; n = 11; Figure 3G, Table 2). In summary, both ESL and S-Lic also inhibited the persistent Na+ current.
3.2 Effect of eslicarbazepine acetate and S-licarbazepine on voltage dependence of activation and inactivation
We next investigated the effect of ESL (300 μM) and S-Lic (300 μM) on the I-V relationship in MDA-MB-231 and HEK-Nav1.5 cells. A Vh of −120 mV was used for subsequent analyses to ensure that the elicited currents were sufficiently large for analysis of kinetics and voltage dependence, particularly for MDA-MB-231 cells, which display smaller peak Na+ currents (Tables 1, 2). Neither ESL nor S-Lic had any effect on the threshold voltage for activation (Figure 4A-D; Tables 1, 2). ESL also had no effect on the voltage at current peak in either cell line (Figure 4A-D; Tables 1, 2). Although S-Lic had no effect on voltage at current peak in MDA-MB-231 cells, it significantly hyperpolarised in HEK-Nav1.5 cells from −18.0 ± 4.2 mV to −30.0 ± 5.6 mV (P < 0.001; n = 9; paired t test; Figure 4A-D; Tables 1, 2).
ESL had no significant effect on the half-activation voltage (V½) or slope factor (k) for activation in MDA-MB-231 cells (Figure 5A; Table 1). The activation k in HEK-Nav1.5 cells was also unchanged but the activation V½ was significantly hyperpolarised by ESL from −39.4 ± 1.3 to −44.2 ± 1.8 mV (P < 0.05; n = 10; paired t test; Figure 5B; Table 1). S-Lic also had no significant effect on the activation V½ or k in MDA-MB-231 cells (Figure 5C; Table 2). However, the V½ of activation in HEK-Nav1.5 cells was significantly hyperpolarised from −32.8 ± 3.1 mV to −40.5 ± 3.4 mV (P < 0.01; n = 9; paired t test; Figure 5D; Table 2) and k changed from 5.9 ± 0.9 mV to 4.5 ± 1.1 mV (P < 0.05; n = 9; paired t test; Figure 5D; Table 2).
As regards steady-state inactivation, in MDA-MB-231 cells, ESL significantly hyperpolarised the inactivation V½ from −80.6 ± 0.7 mV to −86.7 ± 1.2 mV (P < 0.001; n = 13; paired t test) without affecting inactivation k (Figure 5A; Table 1). ESL also hyperpolarised the inactivation V½ in HEK-Nav1.5 cells from −78.2 ± 2.5 mV to −88.3 ± 2.7 mV (P < 0.001; n = 10; paired t test), and changed the inactivation k from −6.9 ± 0.4 mV to −9.8 ± 0.7 mV (P < 0.001; n = 10; paired t test; Figure 5B; Table 1). S-Lic also significantly hyperpolarised the inactivation V½ in MDA-MB-231 cells from - 71.8 ± 2.5 mV to −76.8 ± 2.2 mV (P < 0.05; n = 7; paired t test) without affecting inactivation k (Figure 5C; Table 2). However, the inactivation V½ in HEK-Nav1.5 cells was not significantly altered by S-Lic, although the inactivation k significantly changed from −6.5 ± 0.4 mV to −8.1 ± 0.5 mV (P < 0.05; n = 9; paired t test; Figure 5D; Table 2). In summary, both ESL and S-Lic affected various aspects of the voltage dependence characteristics of Nav1.5 in MDA-MB-231 and HEK-Nav1.5 cells, predominantly hyperpolarising the voltage dependence of inactivation.
3.3 Effect of eslicarbazepine acetate and S-licarbazepine on activation and inactivation kinetics
We next studied the effect of both compounds on kinetics of activation and inactivation. In MDA-MB-231 cells, ESL significantly accelerated the time to peak current (Tp) upon depolarisation from −120 mV to −10 mV from 2.1 ± 0.2 ms to 1.9 ± 0.2 ms (P < 0.01; n = 13; paired t test; Table 1). However, in HEK-Nav1.5 cells, ESL significantly slowed Tp from 1.4 ± 0.2 ms to 1.5 ± 0.2 ms (P < 0.001; n = 14; paired t test; Table 1). S-Lic had no significant effect on Tp in MDA-MB-231 cells but significantly slowed Tp in HEK-Nav1.5 cells from 1.8 ± 0.5 ms to 2.3 ± 0.6 ms (P < 0.01; n = 13; paired t test; Table 2).
To study effects on inactivation kinetics, the current decay following depolarisation from −120 mV to −10 mV was fitted to a double exponential function to derive fast and slow time constants of inactivation (τf and τs). Neither ESL nor S-Lic had any significant effect on τf or τs in MDA-MB-231 cells (Tables 1, 2). However, in HEK-Nav1.5 cells, ESL significantly slowed τf from 0.9 ± 0.1 ms to 1.2 ± 0.1 ms (P < 0.001; n = 12; paired t test; Table 1) and slowed τs from 6.6 ± 0.8 ms to 20.8 ± 8.5 ms, although this was not statistically significant. S-Lic significantly slowed τf from 1.0 ± 0.04 ms to 1.3 ± 0.06 ms (P < 0.001; n = 11; paired t test; Table 2) and τs from 6.3 ± 0.5 ms to 7.3 ± 0.5 ms (P < 0.05; n = 11; paired t test; Table 2). In summary, both ESL and S-Lic elicited various effects on kinetics in MDA-MB-231 and HEK-Nav1.5 cells, predominantly slowing activation and inactivation.
3.4 Use-dependent effect of eslicarbazepine acetate and S-licarbazepine on Nav1.5
To study use-dependent block of Na+ current by ESL and S-Lic, we ran a protocol where cells were subjected to repeated depolarisations from −120 mV to 0 mV, at a frequency of 50 Hz. In MDA-MB-231 cells, this depolarisation train caused a rapid decrease in current size, which reached a plateau of 88.8 ± 2.3 % of the initial current (Figure 6A). In the presence of ESL, the decrease in current reached a plateau of 81.5 ± 2.3 %, suggesting use-dependent block of the channel, although this was not statistically significant (P = 0.10; n = 9; paired t test; Figure 6A). S-Lic had a similar but less obvious effect where the current declined to 80.5 ± 3.1 % in the presence of S-Lic, compared to 86.5 ± 3.1 % without drug (P = 0.31; n = 7; paired t test; Figure 6B).
Use-dependent block was easier to study in HEK-Nav1.5 cells due to the larger Na+ current. In these cells, ESL increased the reduction in current amplitude to 60.6 ± 7.9 % of the initial current, compared to 90.0 ± 2.1 % without drug (P < 0.01; n = 9; paired t test; Figure 6C). S-Lic also increased the reduction in current amplitude to 76.1 ± 2.1 % of the initial current, compared to 85.7 ± 2.2 % without drug (P < 0.001; n = 9; paired t test; Figure 6D). Together, these results indicate that both ESL and S-Lic cause use-dependent block of Nav1.5 at a stimulation frequency of 50 Hz.
3.5 Effect of eslicarbazepine acetate and S-licarbazepine on recovery from fast inactivation
To investigate the effect of ESL and S-Lic on channel recovery from fast inactivation, we subjected cells to two depolarisations from Vh of −120 mV to 0 mV, changing the interval between these in which the channels were held at −120 mV to facilitate recovery. Significance was determined by fitting a single exponential curve to the normalised current/time relationship and calculating the time constant (τr). In MDA-MB-231 cells, ESL significantly slowed τr from 6.0 ± 0.5 ms to 8.7 ± 0.7 ms (P < 0.05; n = 10; paired t test; Figure 7A, Table 1). Similarly, in HEK-Nav1.5 cells, ESL significantly slowed τr from 4.5 ± 0.4 ms to 7.1 ± 0.6 ms (P < 0.001; n = 10; paired t test; Figure 7B, Table 1). S-Lic also significantly slowed τr in MDA-MB-231 cells from 6.8 ± 0.4 ms to 13.5 ± 1.0 ms (P < 0.01; n = 7; paired t test; Figure 7C, Table 2). Finally, S-Lic also significantly slowed τr in HEK-Nav1.5 cells from 5.7 ± 0.7 ms to 8.0 ± 1.2 ms (P < 0.01; n = 10; paired t test; Figure 7D, Table 2). In summary, both ESL and S-Lic slowed recovery from fast inactivation of Nav1.5.
4 Discussion
In this study, we have shown that ESL and its active metabolite S-Lic (both at 300 μM) inhibit the transient and persistent components of Na+ current carried by Nav1.5. We show broadly similar effects in MDA-MB-231 cells, which express endogenous Nav1.5 (29, 30, 45), and in HEK-293 cells over-expressing Nav1.5. Notably, both compounds were more effective when Vh was set to −80 mV than at −120 mV, suggestive of depolarised state-dependent binding. In addition, the inhibitory effect of ESL was reversible whereas inhibition by S-Lic was not. As regards voltage-dependence, both ESL and S-Lic shifted activation and steady-state inactivation curves, to varying extents in the two cell lines, in the direction of more negative voltages. ESL and S-Lic had various effects on activation and inactivation kinetics, generally slowing the rate of inactivation. Both ESL and S-Lic also caused use-dependent block of Nav1.5, although the effect was more obvious in HEK-Nav1.5 cells due to the larger Na+ current. Finally, recovery from fast inactivation of Nav1.5 was significantly slowed by both ESL and S-Lic.
To our knowledge, this is the first time that the effects of ESL and S-Lic have specifically been tested on the Nav1.5 isoform. A strength of this study is that both the prodrug (ESL) and the active metabolite (S-Lic) were tested using two independent cell lines, one endogenously expressing Nav1.5, the other stably over-expressing Nav1.5. MDA-MB-231 cells also express Nav1.7, although this isoform is estimated to be responsible for only ~9 % of the total VGSC current (30, 45). MDA-MB-231 cells also express endogenous β1, β2 and β4 subunits (47–49). MDA-MB-231 cells predominantly express the developmentally regulated ‘neonatal’ Nav1.5 splice variant, which differs from the ‘adult’ variant over-expressed in the HEK-Nav1.5 cells by seven amino acids located in the extracellular linker between transmembrane segments 3 and 4 of domain 1 (30, 42, 45). Notably, however, there were no consistent differences in effect of either ESL or S-Lic between the MDA-MB-231 and HEK-Nav1.5 cells, suggesting that the neonatal vs. adult splicing event, and/or expression of endogenous β subunits, does not impact on sensitivity of Nav1.5 to these compounds. This finding contrasts another report showing different sensitivity of the neonatal and adult Nav1.5 splice variants to the amide local anaesthetics lidocaine and levobupivacaine (44). Our findings suggest that the inhibitory effect of S-Lic on Nav1.5 is less reversible than that of ESL. This may be explained by the differing chemical structures of the two molecules possibly enabling S-Lic to bind the target with higher affinity than ESL. Most VGSC-targeting anticonvulsants, including phenytoin, lamotrigine and carbamazepine, block the pore by binding via aromatic-aromatic interaction to a tyrosine and phenylalanine located in the S6 helix of domain 4 (50). However, S-Lic has been proposed to bind to a different site given that it was found to block the pore predominantly during slow inactivation (10). Alternatively, the hydroxyl group present on S-Lic (but not ESL) may become deprotonated, potentially trapping it in the cytoplasm.
This study used a single concentration for both compounds (300 μM) and the findings presented here broadly agree with in vitro concentrations used elsewhere to study effects of ESL and S-Lic on Na+ currents. For example, using a Vh of −80 mV, 300 μM ESL was shown to inhibit peak Na+ current by 50 % in N1E-115 neuroblastoma cells expressing Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7 (20). S-Lic (250 μM) also blocks peak Na+ current by ~50 % in the same cell line (10). In addition, S-Lic (300 μM) reduces persistent Na+ current by ~25 % in acutely isolated murine hippocampal CA1 neurons expressing Nav1.1, Nav1.2 and Nav1.6 (21–24). Similar to the present study, ESL was shown to hyperpolarise the voltage-dependence of steady-state inactivation in N1E-115 cells (20). On the other hand, similar to our finding in HEK-Nav1.5 cells, S-Lic has no effect on steady-state inactivation in N1E-115 cells (10). Again, in agreement with our own findings for Nav1.5, S-Lic slows recovery from inactivation and causes use-dependent inhibition in N1E-115 cells (10). It should be noted that the frequency of stimulation used in the protocol to assess use-dependent block (50 Hz) is much faster than the typical human heart rate (1-2 Hz), thus, Nav1.5 in cardiac tissue would not be expected to experience use-dependent block to this extent. Nonetheless, these observations suggest that the sensitivity of Nav1.5 to ESL and S-Lic is broadly similar to that reported for neuronal VGSCs. In support of this, Nav1.5 shares the same conserved residues proposed for Nav1.2 to interact with ESL (Figure 8) (51).
Notably, the concentration used in this study is high compared with concentrations achieved in clinical use (e.g. ESL 1200 mg QD gives a peak plasma concentration of ~90 μM) (10). However, it has been argued that the relatively high concentrations required for channel inhibition in vitro are clinically relevant given that S-Lic has a high (50:1) lipid:water partition co-efficient and thus would be expected to reside predominantly in the tissue membrane fraction in vivo (15). Future work investigating the dose-dependent effects of ESL and S-Lic would be useful to resolve these possibilities and aid clinical judgements.
The data presented here raise several implications for clinicians. The observed tonic and use dependent inhibition of Nav1.5 is worthy of note when considering cardiac function in patients receiving ESL (13). Although the QT interval remains unchanged for individuals on ESL treatment, prolongation of the PR interval has been observed (27). Further work is required to establish whether the basis for this PR prolongation is indeed via Nav1.5 inhibition. In addition, it would be of interest to investigate the efficacy of ESL and S-Lic in the context of heritable arrhythmogenic mutations in SCN5A, as well as the possible involvement of the β subunits (24, 26, 52, 53). The findings presented here are also relevant in the context of Nav1.5 expression in carcinoma cells (54). Given that cancer cells have a relatively depolarised Vm, it is likely that Nav1.5 is mainly in the inactivated state with the persistent Na+ current being functionally predominant (55, 56). Increasing evidence suggests that persistent Na+ current carried by Nav1.5 in cancer cells contributes to invasion and several studies have shown that other VGSC inhibitors reduce metastasis in preclinical models (29–35, 57). Thus, use-dependent inhibition by ESL would ensure that channels in malignant cells are particularly targeted, raising the possibility that it could be used as an anti-metastatic agent (43). This study therefore paves the way for future investigations into ESL and S-Lic as potential invasion inhibitors.
5 Author Contributions
TL, SC and WB contributed to the conception and design of the work. TL, LB and WB contributed to acquisition, analysis, and interpretation of data for the work. TL, SC and WB contributed to drafting the work and revising it critically for important intellectual content. All authors approved the final version of the manuscript.
7 Acknowledgements
This work was supported by Cancer Research UK (A25922) and Breast Cancer Now (2015NovPhD572).
8 Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
9 Data availability statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
6 Abbreviations
- ESL
- eslicarbazepine acetate
- HEK-Nav1.5
- HEK-293 cells stably expressing Nav1.5
- I-V
- current-voltage
- k
- slope factor
- PSS
- physiological saline solution
- S-Lic
- S-licarbazepine
- Tp
- time to peak current
- τf
- fast time constant of inactivation
- τs
- slow time constant of inactivation
- τr
- time constant of recovery from inactivation
- VGSC
- voltage-gated Na+ channel
- Vm
- membrane potential
- Vh
- holding potential
- Vpeak
- voltage at which current was maximal
- Vrev
- reversal potential
- Vthres
- threshold voltage for activation
- V1/2
- half-activation voltage