Experimental evidence gathered during the past 15 to 20 years suggests that the sodium channel is a primary target for a variety of AEDs, the most effective of which are phenytoin (PHT), CBZ, and LTG where sodium channel blockade is considered to be the main mechanism of action (Table 1). Additionally, a host of other AEDs, including new and potential AEDs, whose main mechanism of action is often unrelated to sodium currents, have been shown to interact with voltage-gated sodium currents or INa-dependent processes. Some of these reports merit a note of caution: Whereas voltage-clamp experiments on mammalian neurons can specifically disclose sodium current alterations in nerve cells, analysis of spike shape, repetitive firing properties, toxin binding, or sodium uptake only indirectly reflects sodium channel involvement. Thus action-potential shape and repetitive firing properties are critically determined not only by sodium inward, but also by several outward currents as well. Drugs affecting potassium currents, for example, will automatically cause changes in these parameters. Studies in nonmammalian expression systems such as Xenopus oocytes may be difficult to interpret (see section on ion channels). Last, some drugs were shown to have effects on sodium currents or other sodium current–dependent processes, but at concentrations beyond therapeutic levels, and thus the mechanism of action cannot be considered to consist of sodium current modulation. For these reasons, Table 1 summarizes the data obtained in voltage-clamp experiments on mammalian neurons. It reveals that of the drugs discussed later, only CBZ, PHT, LTG, and felbamate (FBM) have been demonstrated to affect sodium currents specifically, with ambiguous data on VPA and topiramate (TPM). For the sake of completeness, all other substances are discussed as well. Special emphasis is given to voltage-clamp experiments whenever available, which are listed first in the different sections.
Actions of anticonvulsants on INa
Direct evidence of an interaction of CBZ with voltage-gated sodium currents has been gathered in a variety of neuronal preparations, and CBZ may share an extracellular binding site in sodium channels with PHT and LTG, whose actions on INa are discussed later (152). In accutely isolated hippocampal neurons, obtained during epilepsy surgery from patients with intractable epilepsy, or from rats, CBZ reduces sodium inward currents (98,137,153). This effect is primarily caused by a shift of the steady-state inactivation curve to hyperpolarizing potentials, thereby reducing availability of sodium channels at resting membrane potential levels, whereas CBZ does not modify activation parameters (98,137,153). The suppression of INa is very much voltage dependent [i.e., the effect was pronounced at depolarized membrane potential, with the apparent median inhibitory concentration (IC50) of 900 μM when eliciting currents from –100-mV holding potential reduced to 20 μM when stepping from –70 mV, indicating that CBZ binds primarily to the inactivated sodium channel](153). In comparison to PHT tested in the same preparation (see later), CBZ block occurs ∼5 times faster, but with lower affinity, suggesting that antiepileptic effects might be expected to be more pronounced with short-duration discharges (137,153). Additionally, CBZ is less potent in shifting the steady-state inactivation curve in neurons from chronically epileptic rats (98). A blocking effect of CBZ on INa also was reported for neuroblastoma cells maintained in culture. CBZ reduced sodium currents with half-maximal inhibition at ∼30–140 μM, and the block was both voltage and use dependent (i.e., it increased with repetitive stimulations) (154–156). Investigations in rat dorsal root ganglion neurons suggest that the action of CBZ may be dependent on the sodium channel subtype. Thus action-potential amplitudes as well as sodium currents were reduced potently by CBZ at a concentration of 100 μM in cells that displayed TTX-sensitive currents, whereas in TTX-resistant neurons, even CBZ concentrations of 300 μM were much less effective (157). This appears to hold particularly true for TTX-resistant currents displaying steady-state inactivation only at relatively positive potentials (158) and may explain why no effect of CBZ on sodium currents in dorsal root ganglion cells has been reported when the substance was used at a low concentration of 50 μM(159). It is interesting to note a similarity of findings in dorsal root ganglion cells (158) and CA1 neurons from kindled rats (97,98). Both show inactivation at relative depolarized potentials, and CBZ in these neurons is less effective in modulating INa.
Other evidence of a suppressive action of CBZ on INa was obtained in mammalian cell line transfected with Nav1.2 (160), mammalian and amphibian nerve fibers (161,162), and snail neurons (163), in essence confirming a voltage- and use-dependent block and negative shift of the steady-state inactivation curve. The reduction of sodium current under voltage-clamp conditions is reflected in the amplitude reduction of action potentials, and more important, a suppression of high-frequency repetitive firing as a functional consequence of prolonged binding of CBZ to sodium channels. This has been confirmed in both nerve fibers and neurons (165–167). At least at low concentrations of 30 μM, regular synaptic transmission, however, as judged by the effect of CBZ on evoked field potentials, is not impeded (168). The interaction of CBZ with sodium channels has been investigated by studying the competition of CBZ with binding of batrachotoxinin (BTX), a molecule adhering specifically to a site on the channel molecule responsible for the activation of sodium current, and by competition with veratridine or veratrine, two sodium channel openers. CBZ displaces BTX binding and reduces veratridine effects in rat brain synaptosomes and neocortical slices (168–171). Furthermore, it may act by modulating synaptic transmission due to blockade of sodium influx, as measured by 22Na+ uptake in synaptosomes or adrenal medullary cells homologous with sympathetic postganglionic neurons (172,173).
Although its main mechanism of action is generally thought to involve the γ-aminobutyric acid (GABA)ergic inhibitory system, some experiments suggest sodium current blockade as one effect of DZP. In isolated rat hippocampal neurons, as well as in mouse neuroblastoma cells, DZP reduces INa by 40% from a holding potential of –80 mV, however, at concentrations way beyond therapeutic levels. This reduction is voltage dependent and caused by a negative shift of the inactivation curve, which appears to be less pronounced than the one obtained with PHT and CBZ (154,174).
DZP, in nanomolar concentrations, reduces repetitive firing in spinal cord neurons in culture, as do nitrazepam (NZP), lorazepam (LZP), and clonazepam (CZP) (175). In higher concentrations of 100 μM, DZP reduces compound action potentials in rat optic nerves, suggesting that, as for other AEDs, repetitive firing is much more vulnerable to DZP than are single action potentials (166).
ESM, commonly known as a T-type calcium channel blocker, appears to block sodium currents in some preparations. In squid axons, ESM reduces sodium peak currents, although at very large and clinically irrelevant concentrations of 60 mM(177). Interestingly, the block is voltage dependent when applied internally, and voltage independent when perfused externally, suggesting two different blocking mechanisms, the former of which should primarily influence repetitive firing (177). In cat and rat thalamic neurons, ESM was reported to suppress primarily INaP without affecting INa(178) (see earlier). Both T-type calcium currents, as well as INaP, have been speculated to be especially suited to convey burst firing in neurons, a firing pattern deemed to pace epileptic spike-and-wave discharges during absence seizures by thalamocortical interplay. Correspondingly, ESM does reduce burst firing in thalamocortical neurons (178).
In isolated rat striatal neurons, FBM reduced INa with an IC50 of 28 μM, inducing (like other AEDs) a shift of the inactivation curve to negative potentials (179).
Repetitive firing was likewise attenuated, although the first action potential remained unchanged, as were evoked excitatory postsynaptic or field potentials in striatal slice preparations (179). In a voltage-clamp study on Xenopus oocytes in which human or rat brain sodium channels had been expressed, FBM was effective in reducing INa only when applied intracellularly in a high concentration of 1 mM, again inducing a negative shift of the inactivation curve (180). FBM has been reported to reduce repetitive firing in cultured mouse spinal cord neurons (181).
GBP, which displays interactions particularly with synaptic systems, also may act by modulation of INa, but the mechanisms of this action appear to be less straightforward than those with other AEDs. Thus GBP reduces repetitive firing in mouse central neurons in culture, but this effect strongly depends on the duration of exposure to the drug and is maximal only after 12–48 h of incubation (176). Strikingly, the IC50 decreases substantially from ∼130 to 4 μM when the exposure time is increased from 60 s to 48 h (176), suggesting that an effect on sodium channels is exerted only after slow uptake or by metabolic channel modifications. This exceedingly long latency required to show full efficacy may explain why generally negative findings were reported for GBP. Thus with shorter application times or lower concentrations, GBP did not block repetitive firing in neuronal cultures of spinal cord (182), nor INa in cell lines expressing rat brain sodium channel (183) or snail neurons (184). In line with the notion that GBP does not bind directly to sodium channels is the observation that GBP does not reduce BTX binding (185).
The interactions of LTG with sodium channels appear to be very similar to those found for PHT. Thus in voltage-clamp studies on neurons, accutely isolated or in culture, or neuroblastoma cells in culture, LTG potently blocked INa(32,155,186–189)(Fig. 3). This action was strongly voltage and use dependent, with IC50 ranging from 500 to 60 μM at holding potentials of –90 to –60 mV and ∼20% reduction of INa at concentrations as low as 1 μM(32,155,186–188), and associated with a negative shift of the steady-state inactivation curve (32,187–189) and a prolonged recovery from inactivation (Fig. 3)(31,32), reducing the availability of sodium channels and suggesting a block, particularly in the inactivated state. Like PHT, LTG is suggested to bind to sodium channels relatively slowly (187).
Repetitive firing was found to be reduced by LTG in concentrations of 10 to 100 μM in hippocampal and spinal cord neurons, without effects on single action potentials (190,191). Furthermore, LTG inhibits BTX binding in rat brain synaptosomes, as well as veratrine- or veratridine-induced release of NO and glutamate in rat brain both in vivo and in vitro (169,192,193).
Data on the mechanism of action of LSG are scarce. Two reports support the notion that LSG may modulate INa. Thus LSG reduced the frequency of spontaneously occurring action potentials in cultured hippocampal neurons, as well as repetitive firing in the same preparation (194), confirming that LSG increases spike frequency habituation in entorhinal cortex neurons (195).
OCBZ, which structurally only differs slightly from CBZ, and which is rapidly metabolized to an active 10-monohydroxy compound (MHD), effectively blocks repetitive firing of spinal cord neurons in culture, as does MHD. The IC50 for this effect was very low for both drugs: 50 nM for OCBZ, and 20 nM for MHD (196). Furthermore, like CBZ, OCBZ and MHD appear to act frequency dependently (i.e., with increasing stimulation frequencies, action potentials are elicited with decreasing probability) (196).
Phenobarbital (PB) and pentobarbital (PTB).
Evidence that barbiturates—generally acting on the GABAergic system—like PB or PTB might influence INa is scarce and is derived primarily from experiments on amphibian nerve fibers as well as squid and mammalian axons. Under voltage-clamp conditions, sodium inward currents are completely blocked by 3 mM PTB internally applied in squid axons (i.e., in a concentration nearly two orders of magnitude larger than therapeutic ones) (197). Similar findings have been reported in frog nerve fibers, with an IC50 close to 600 μM with extracellular application (161). Current flowing through human brain sodium channels incorporated into lipid bilayer membranes was voltage dependently diminished by PTB (198). Likewise, PB reduced sodium spikes at concentrations of ∼5 mM, and repetitive firing ∼1 mM, in rat preganglionic sympathetic nerves (165). PTB inhibits veratridine-stimulated 22Na+ uptake into synaptosomes, an effect precluded by prior long-term PB treatment, suggesting that, as with VPA (see later), some kind of adaptive process is initiated by prolonged sodium channel blockade (199).
As for CBZ, there is compelling evidence that PHT acts primarily by reducing INa, with only scanty reports explicitly denying sodium current modulation (200). Thus in neocortical neurons from rats, PHT in concentrations as low as 1 μM diminishes sodium currents by ∼20%, with an IC50 of 50 μM(187,201). Again, as with CBZ, this effect is use dependent and caused by a shift of the voltage dependence of inactivation in the hyperpolarizing direction (187,201). The effects of PHT on sodium currents have been studied very extensively in hippocampal neurons and neuroblastoma cell lines, yielding subtle differences in PHT action as compared with that of CBZ. Although the effect of PHT is equally voltage dependent, with IC50 ranging from 600 μM to <10 μM, in a holding-potential range of –90 to –50 mV (153–156,174,202,203), binding of CBZ is much slower and requires long depolarizations (up to seconds) to produce blocking effects (153,202,203) (see earlier). Consistent with this observation, single channel analysis revealed that late channel openings, rather than those immediately after the voltage step, are preferentially diminished by PHT (204,205), which may explain why plateau depolarizations underlying bursts as well as the persistent sodium current INaP are blocked, whereas action-potential generation is left intact (see later) (86,203,204). Different sensitivities of TTX-sensitive and TTX-resistant neurons, as have been observed for CBZ, also are present with PHT (157,158).
Blocking effects of PHT on INa were likewise reported in other preparations, such as Xenopus oocytes in which human brain sodium channels were expressed (205), a mammalian cell line transfected with rat Nav 1.2 (160), and nerve fibers (161,162,209). As functional consequences of slow sodium channel blockade, PHT limits repetitive firing without affecting single action potentials in neurons or evoked potentials in neuronal populations, at least in lower concentrations (154,166,168,210,211). Like CBZ, PHT itself binds to and reduces BTX binding to these channels (170,171,212,213). Mutations of the rat SCN2a sodium channel suggest that PHT binds to the inner lumen of the channel pore and shares this binding site with lidocaine and other local anesthetic drugs (150). Furthermore, PHT, like CBZ, reduces effects of the sodium channel opener veratridine (169,214,215).
In hippocampal slice preparations, REM, as well as its metabolite ARL12495AA reduces sustained repetitive firing of single neurons, with their IC50 values being practically identical at ∼60 μM(216). Furthermore, they also reduce action-potential amplitudes in higher concentrations of 120–400 μM(216). Regarding the racemic mixture of the metabolite, the respective stereoisomers are equally effective (217). Even lower IC50 values for the reduction of repetitive firing were reported for mouse spinal cord neurons in culture (i.e., 8 μM for REM, and 1 μM for its metabolite FPL12495AA), with the effect being voltage and use dependent (218).
Regarding STM, only one report on isolated hippocampal neurons confirms an action on INa(219). In this study, both peak current amplitudes and repetitive spiking were reduced by STM in concentrations of 35 μM.
The action of TPM on INa does not appear to consist of a straightforward block in all preparations: In rat cerebellar granule cells in culture, TPM was effective and depressed INa with a shift of the inactivation curve to hyperpolarized potentials with an IC50 of 50 μM(220).
Furthermore, TPM has been reported to attenuate repetitive firing in hippocampal and spinal cord neurons (221–223), although possibly with a distinctly delayed or even only transient action (210). Correspondingly, extracellular field potentials in hippocampal slices were decreased by TPM in a frequency-dependent fashion (223). In isolated neocortical neurons, however, TPM was reported to be ineffective in blocking INa at low concentrations (25–30 μM), and only at 100 μM did it cause a reduction associated with a left shift of the inactivation curve.
In rat neocortical neurons in cell culture, extracellularly applied VPA (0.2–2 mM) reduced sodium currents by ∼25%(224). The same holds true for human hippocampal neurons, which were obtained during epilepsy surgery; despite the therapy resistance of the patients, VPA led to a negative shift of the steady-state inactivation curve of INa, thus reducing excitability, with an IC50 of 1.4 mM(137). Similar results were obtained in hippocampal neurons of kindled and sham-operated rats (98).
In voltage-clamp experiments on axons of the squid, VPA led to a slowing of the activation of sodium currents and to a voltage-dependent reduction of peak sodium currents, notably only with internal application, suggesting an intracellular site of action (177). Likewise, VPA reduces INa in frog nerve fibers (225) and axons of the annelid Myxicola, shifting the inactivation curve to hyperpolarized potentials (226,227). The notion that VPA exerts its antiepileptic effect by sodium channel blockade is not unchallenged. In one study on hippocampal slices, evoked repetitive (doublet) firing was only slightly affected at physiologic temperatures, and an effect became evident only at <30°C (228). Paradoxic reactions have been reported as well. Prolonged treatment with VPA leads to an increase in sodium channel density and veratridine-induced current (229). Matching these data, in Helix neurons, Altrup et al. (230) observed a decrease and successive reincrease of action-potential duration with extracellular application of VPA (10 mM), and indeed, in the sodium current–dependent veratridine epilepsy model, VPA was reported to be proepileptic (231). The biphasic action of VPA as seen in the snail experiments was shown to be dependent on VPA first acting extracellularly, and then intracellularly (230). Apparently VPA slowly diffused into the cells, and only there, according to the authors, it exerts its main long-term antiepileptic effect, mediated in this case through potassium current modulation. An intracellular action, together with slow diffusion into the cells, would explain the delay of VPA action observed in clinical practice.
The action of ZNS on sodium currents has been investigated only in annelid (Myxicola) axons. In this preparation, the compound reduces INa, with a concentration as low as 12 μM yielding half-maximal effects, which consist mainly, as for other AEDs, in a negative shift of the inactivation curve (227).
Apart from the previously mentioned AEDs, a host of other compounds display anticonvulsant properties in experimental epilepsy models, and at the same time modify sodium current or sodium current–dependent processes such as repetitive neuronal firing. To list these findings extensively would go beyond the scope of this review. For the sake of completeness, some of the drug classes should, however, be mentioned. Among them are, not surprisingly, local anesthetic drugs such as lidocaine, whose prime action is considered to be exerted on INa(232), unspecific calcium channel modulators such as flunarizine, which also act on sodium channels (233,234), kava pyrones (235,236), aconitines (237,238), the candidate AED ralitoline (239,240), and the neuroprotective drug riluzole (187,241–244). In light of a renaissance of the ketogenic diet, it is of interest to note that polyunsaturated fatty acids (abundantly found in this diet), like AEDs discussed earlier, also cause a left shift of the inactivation curve of INa in micromolar concentrations (245).
Actions of anticonvulsants on INaP
As already stated, the persistent sodium current lacks the safety factor of inactivation the fast current possesses. Further, it is active also at resting membrane potential and does not need activation by depolarization. Thus small changes of INaP may have profound effects on neuronal excitability. On the basis of these findings, in addition to a large number of investigations on INa (see earlier), some studies have been carried out to test the effect of AEDs explicitly on INaP. In these studies, INaP was usually depressed by the AEDs. Thus PHT (50–200 μM) was shown to reduce neuronal excitability by blocking repetitive firing (207) and strongly diminishing INaP both in isolated cells and neurons in neocortical slices (206,207) and in isolated hippocampal neurons in microcultures (205). In lower concentrations of 8 μM, persistent TTX-sensitive plateau potentials were likewise blocked, whereas repetitive firing remained unaffected, suggesting that PHT reduces INaP, while sufficiently many sodium channels can still be recruited to generate action potentials (86). In neurons of the snail Lymnaea stagnalis, INaP induced by the convulsant PTZ was equally reduced by CBZ, although in rather high and clinically irrelevant concentrations of 1 mM(164). VPA (10–100 μMl) likewise decreased repetitive neuronal firing and epileptiform activity in rat amygdala slices and abolished the inwardly rectifying response to depolarizing current injection brought about by INaP(246). Likewise, in neocortical neurons, VPA was reported to block INaP selectively at concentrations of 10–30 μM(247). TPM showed some depressive effect on INaP in this preparation, which was, however, much less pronounced (248). ESM (750–1,000 μM) decreased INaP in thalamic neurons, notably without affecting the fast sodium current (178).