Sei sulla pagina 1di 16

Current Topics in Medicinal Chemistry 2005, 5, 15-30

15

Voltage Gated ion Channels: Targets for Anticonvulsant Drugs

Adam C. Errington 1,3 , Thomas Stöhr 2 and George Lees 1,3,*

1 Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin, New Zealand, 2 Schwarz Biosciences GmBH, Alfred Nobel Strasse 10, 40789 Monheim am Rhein, Germany,

3 Sunderland Pharmacy School, University of Sunderland, Chester Road Campus, Sunderland SR1 3SD, UK

Abstract: Epilepsy is one of the most prevalent neurological syndromes in the world today. Epilepsy describes a group of brain disorders whose symptoms and causes are diverse and complicated, but all share a common behavioural manifestation: the seizure. Seizures result from the abnormal discharge of groups of neurons within the brain, usually within a focal point, that can result in the recruitment of large brain regions into epileptiform activity. Although the range of explanations for the development of seizures can be as varied as genetic composition to acute head trauma, the net result is often similar. The excitability of neurons is governed by the input they receive from their neighbours and the intrinsic excitability of the neuron. In this review we focus on elements that are crucial to determining the intrinsic excitability of neurons in the CNS, the voltage gated ion channels (VGICs).

VGICs as well as being important for physiological function are critical in producing hyperexcitability such as that associated with seizure discharges. Many drugs routinely used in the clinical setting, as well as several novel experimental drugs, have shown interactions with VGICs that underpin, at least in part, their anticonvulsant action. We review the physiological roles of voltage gated ion channels that are selective for sodium, potassium and calcium conductance and attempt to highlight their role in the pathology of epilepsy. This is supplemented by the mechanisms of drug action at these important anticonvulsant targets for classical and clinically relevant compounds (e.g. phenytoin, ethosuximide) as well as some important second generation drugs (e.g. gabapentin, levetiracetam) and novel experimental agents (e.g. retigabine, losigamone, safinamide). We also briefly discuss the urgent need for new drugs in this arena and the potential of combinatorial methods and recombinant screening to identify leads.

Key Words: Voltage gated ion channels; epilepsy; anticonvulsants

INTRODUCTION

Epilepsy is one of the most prevalent neurological disorders with current estimates approximating between 0.5 – 2% of the global population being affected. The use of the term epilepsy is to some extent misleading as it actually refers to a collection of neurophysiological disorders that are diverse in terms of both their etiology and symptomology. The typical behavioural manifestation common to all forms of epilepsy is the aberrant synchronized discharge of neuronal populations termed the ‘seizure’. Seizures result from phasic changes in the firing properties of groups of neurons, usually within a discrete focal point, to an intermittent high-frequency burst-firing mode. Induction of a population of neurons into a pattern of burst-firing resulting in synchronized disruptive seizure discharges is determined by changes in excitability of neurons that are essentially governed by two major factors. The first determinant of neuronal excitability in any given neuron, is the balance of synaptic input from neighboring cells within the network in the form of chemical transmission (through ligand gated ion channels and G-protein coupled metabotropic receptors) and aplastic but extremely rapid electrical transmission (through gap juctions). Secondly, and perhaps most importantly are

*Address correspondence to this author at Chair & Head of Department of Pharmacology & Toxicology, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin, New Zealand; Tel: 64 3 4797265; Fax: 64 3 4799777; E-mail: george.lees@stonebow.otago.ac.uk

1568-0266/05 $50.00+.00

the electrogenic properties intrinsic to each neuron. These are determined to a great extent by the complement and localization of functional voltage gated ion channels expressed in the cell membrane [1]. It is intuitive, therefore, that these determinants of physiological excitability would be crucially involved in the pathological hyperexcitability occurring during the seizure discharges characteristic of the epilepsies. Indeed both excitatory and inhibitory neurotrans- mission as well as voltage-gated channels (that can be modulated selectively by natural or synthetic toxins), have been shown to be crucial for seizure like activity in acute chemoconvulsant models in rodent brain slices and in vivo [2]. It is not surprising, therefore, that many of the drugs used in the treatment of seizures and epilepsy target the channels and receptors that are vital for the initiation and spread of seizure activity. In this review we focus specifically upon the voltage gated ion channels, discussing their function both physiologically and pathologically and evaluate their relevance as anticonvulsant drug targets by outlining how they can be manipulated for therapeutic benefit.

ELECTROPHYSIOLOGICAL PROPERTIES AND MOLECULAR BIOLOGY OF VOLTAGE GATED SODIUM CHANNELS (VGSCS)

The action potential is the cellular mechanism by which excitable cells within the nervous system communicate with one another. Action potentials propagating in large

© 2005 Bentham Science Publishers Ltd.

16

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

pyramidal cells of the cortical surface of the brain constitute the unitary events in an EEG. At the cellular level, using the newly developed voltage clamp technique in the 1950s Hodgkin and Huxley showed that a transient voltage activated sodium current (I Na ) was responsible for the rapid rising phase of this crucial intracellular communication process. Although the techniques required to elucidate the molecular correlate for this current were not available in the 1950s these seminal studies identified three essential features that have come to characterize the voltage gated sodium channel (VGSC) family, namely: voltage dependent activation, rapid inactivation and selective ion conductance [3-5]. Following this series of elegant and pioneering experiments a large number of detailed electrophysiological and molecular studies have elucidated much about the structure and function of the channel(s) responsible for I Na . Molecular biology has determined the major structural component of the channel to be a protein of approximately 260 kDa, called the a -subunit, which has four repeated (named I-IV) structural motifs consisting of six a -helical transmembrane spanning domains separated by intra and extracellular loops [6]. These four repeated domains fold together to form a central pore and it is their structural components that determine selectivity and conductance of the ion channel. Although a subunits alone can form functional channels co-expression of accessory b (33 kDa) subunits is most commonly required to confer the correct gating properties found in native channels. Channels formed purely from a subunits exhibit slower inactivation and positive shifts in the voltage dependence of activation compared to native neurons when expressed in Xenopus oocytes. To date three b (b1-3) accessory subunits have been cloned from rat and human and their effects upon channel gating have been extensively characterised

[6-8].

Based upon sequence homology, several different sodium channel a subunits, recently named by IUPHAR as Na V 1.1-

1.9 [9], have been characterised. cDNA clones have been isolated from rat brain cDNA libraries and show that the major central nervous system (CNS) channels are encoded for by four genes entitled Scn1a, Scn2a, Scn3a and Scn8a [9- 11]. The corresponding protein products of these genes (or splice variants of them) form the major brain sodium channel a -subunits classified as Na V 1.1-1.3 which are blocked by the puffer fish ‘fugu’ poison (Tetrodotoxin, TTX). Several mutations of the Scn1a and the Scn2a genes have been identified in individuals suffering from monogenetic epilepsy syndromes such as generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy of infancy (table 1). The gene Scn8a encodes the primarily CNS Na v 1.6 a -subunit (formerly SkM2) and a further CNS channel resistant to TTX, Scn5a (Na v 1.5, formerly NaCh6), has also been described. Crystal structures for these large homo-oligomeric pores remain elusive but the fine structure of the channel complex has been determined by means of cryo-electron microscopy and image reconstruction analysis. This reveals water filled pores (thought to contain the gating charges) deep within the membrane plane and multiple intracellular vestibules which constitute a likely pathway for the permeant ions [12].

Functionally, VGSCs can be simply thought to exist in three conformational states and it is the transition between these states that allows selective and temporally regulated ionic conductance. At the resting potential of most CNS neurons, VGSCs can be found in a closed non-conducting state commonly referred to as the ‘resting’ state (Fig. 1C). In response to change in membrane potential, such as that provided by excitatory synaptic input [13], the channels undergo a physical conformational change. Activation occurs within a fraction of a millisecond and as predicted by Hodgkin & Huxley [14] is due to movement of gating charges within the membrane electric field. These so called ‘gating currents’ have been measured electrophysiologically [15,16] and attributed to sequences of positively charged

Table 1.

Voltage Gated ion Channel Genes Bearing Mutations which have been Linked to Inherited Epilepsy.

Voltage-gated ion channel family

Gene name

Syndrome

Reference

Potassium channels

KCNQ2

BFNC

[113,119]

KCNQ3

BFNC

[119,120]

KCNQ3

JME

[121]

Sodium channels

SCN1A

GEFS+

[122-124]

SCN1A

SMEI

[125,126]

SCN2A

GEFS+

[127]

SCN2A

BFNIC

[128]

SCN1B

GEFS+

[129]

Calcium channels

CACNA1A

IGE

[130]

 

CACNA1H

CAE

[131]

BFNC: Benign familial neonatal convulsions BFNIC: Benign familial neonatal/infantile convulsions CAE: Childhood absence epilepsy GEFS+: Generalized epilepsy with febrile seizures plus IGE: Idiopathic generalised epilepsy JME: Juvenile myoclonic epilepsy SMEI: Severe myoclonic epilepsy of infancy

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

17

Topics in Medicinal Chemistry, 2005 , Vol. 5, No. 1 17 Fig. (1) . (A) Structures

Fig. (1). (A) Structures of anticonvulsant drugs that act upon VGSCs. (B) Diagram of the subunit structure of VGSC a and b subunits showing the pore forming region, the voltage sensor and inactivation gate. (C) Gating conformations of the voltage gated subunit.

arginine and lysine residues in the S4 regions of the a subunit [6] (Fig. 1B). The movement of the S4 region perpendicular to the plane of the membrane results in opening of the activation gate and allows sodium flux. For the VGSC it was estimated that the movement of approximately six charges across the entire membrane would be required for gating [7]. Neutralization of the charged residues in the S4 region markedly alters the voltage dependence of activation [17]. Upon opening of the activation gate, the channel pore (formed by a re-entrant loop between transmembrane segments V and VI) selectively conducts sodium ions along their electrochemical gradient from the extracellular space to the cell interior thus depolarizing further the neuronal transmembrane potential. However, within a few milliseconds of opening of the activation gate, sustained depolarization results in termination of the sodium conductance by a process known as inactivation. Inactivation usually (but not always, as discussed below) occurs rapidly, attenuating the majority of the sodium current and for this reason is often referred to as ‘fast’ inactivation. The process of fast inactivation has been strongly linked to the short intracellular loop between domains III and IV (the so-called III-IV linker) [18,19] (Fig. 1B) which is also known as the inactivation gate. Enzymatically cutting this loop [17], deleting amino acids in the linker [18], or directing antibodies against it [19], all produce a marked slowing or disruption of the inactivation process. It is hypothesised that the intracellular portion of the protein may bind to a so called ‘inactivation gate receptor’ on the cytoplasmic side of the IVS6 domain using a structural motif comprising a hydrophobic group of isoleucine, phenylalanine and methionine (IFM) [20,21] (Fig. 1C). Entry of sodium channels into the fast inactivated-state is highly voltage dependent and becomes significantly more pronounced upon depolarization. The ability of channels to recover from the fast inactivated state is dependent upon

membrane potential and time and is a mechanism that ensures adequate time for recovery before reopening of the channel [22].

The properties of rapid activation and inactivation of VGSCs are essential determinants in neuronal firing frequency. As such they are also crucial for signal transmission in neuronal networks including recruitment of oscillatory activity of putative importance for both cognition and consciousness (i.e. g frequency) [23]. However the ability of sodium channels to rapidly cycle through conform- ational states and facilitate high frequency neuronal firing is also indispensible for the generation of synchronous bursting activity that is characteristic of epilepsy. Indeed computer models of normal and disruptive (epileptic) neuronal network oscillations require the inclusion of the fast transient sodium current (I Na ) carried by neuronal VGSCs [1,23].

As previously stated, sodium channel inactivation is often thought of as a single process but it is increasingly clear that there are at least two distinct kinetic classes of inactivation (ultra-slow activation is discussed later). The second type of sodium channel inactivation, termed slow, is postulated as a cellular back-up mechanism that prevents I Na from becoming fully available rapidly. This type of inactivation appears to be independent of the III-IV linker (crucial for fast inactivation) and has recovery time constants in the order of several hundreds of milliseconds to seconds [22]. Recruitment of VGSCs into the slowly inactivated conformation appears to proceed only under conditions of prolonged or high frequency depolarization [24-26]. Furthermore, there is an apparent proportional relationship between the time taken to recover from the slowly inactivated state and the duration of depolarizing challenge [27]. Type II and IIA sodium channels expressed in the Xenopus oocyte expression system showed the time constant of recovery from slow inactivation was intrinsically related

18

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

to the duration of previous depolarizations by a power law:

t(t) = p t D (where t is the recovery constant for the channel from slow inactivation, t is the duration of previous depolarization, p is a constant kinetic setpoint and D a positive scaling power). This type of inactivation process is especially pertinent to epileptiform activity as the neuronal networks involved are often exposed to periods of prolonged high frequency neuronal activity [28-31] that may be ‘damped down’ by slow inactivation. It appears therefore that pharmacological enhancement or stabilization of the slowly inactivated conformation of VGSCs may be useful in disrupting prolonged neuronal seizure discharges.

Although I Na is the principal sodium current responsible for neuronal activity the existence of a second type of sodium current, termed the persistent current (I NaP ), has been confirmed in a number of neuronal cell types [32]. This current, although only representing 1-3% of the total peak current is thought to be crucial for determination of spike threshold in neurons [32]. The mechanism behind these persistent currents is not clearly defined but has been variously attributed to the failure of a fraction of channels (responsible for conducting I Na ) to inactivate or the ability to switch to an ‘ultra-slow’ inactivating gating conformation [32-35]. The voltage dependence of activation of I NaP differs from that of I Na . Persistent Na + currents are activated at potentials below spike threshold (-60mV) and reach peak at between –40 and –35mV with V 50 for activation at around –50mV [33,36-38]. I NaP has been implicated in the fine control of neuronal excitability based upon the sub-threshold activation kinetics and lack of rapid inactivation. As previously highlighted, synchronous neuronal network activity (like that associated with seizures) is strongly dependent upon the ability of neurons to fire bursts of action potentials. I NaP has been shown to be crucial in both hippocampal CA1 neurons and layer V neocortical neurons in the induction of burst firing modes. Evidence exists that I NaP is also thought to, at least in part, contribute to rhythmic sub-threshold oscillations that can determine behaviour of entire neuronal networks in a number of structures including the neocortex and entorhinal cortex [23]. It appears that I NaP is acting as a mechanism for ‘fine-tuning’ neuronal excitability. Selective modulation of this current may represent an attractive target for future anticonvulsant drug development. It is possible that a ligand selective for I NaP may be able reduce hyperexcitability without the drawbacks associated with attenuating I Na .

A new and exciting putative target for future anticonvulsant drug intervention has come to light relatively recently and is based upon a somewhat unexpected form of plasticity displayed by VGSCs (see refs [7,39] for detailed review). Biochemical experiments including two dimensional phosphopeptide mapping have shown purified sodium channel a subunits have five distinct phosphorylation sites throughout the glycopeptide. Four of these sites are found on serine residues (573, 610, 623 and 687) in the large intracellular loop connecting domains I and II (L I-II ) and a further one is located of the domain III-IV linker which constitutes the inactivation gate [40]. In intact synaptosomes the Na + flux induced by veratridine is reduced by circa 26% when activators of cAMP dependent protein kinase (PKA) are concurrently applied due to rapid phosphorylation of a

subunits. In functional studies activation of PKA resulted in a 25-40% reduction in peak Na + current mediated by Na V 1.2 channels expressed in Xenopus oocytes [41] or CNaIIA-1 [42] (CHO cell derived stable line expressing Na V 1.2) cells without shifting voltage dependence of activation or inactivation.

To complement this evidence it has also been shown that protein kinase C (PKC) is able to phosphorylate sodium channel a subunits. The amplitude of sodium current is reduced and the rate of inactivation slowed in neuroblastoma treated with fatty acids such as OAG (1-oleoyl-2-acetyl-sn- glycerol) or DOG (1,2-dioctanoyl-rac-glycerol) that activate PKC by mimicking the endogenous second messenger molecule DAG. Slowed inactivation of sodium channels produced by activation of PKC results from phosphorylation of the site situated on the inactivation gate that does not appear to be targeted by PKA. In Xenopus oocytes transiently expressing rat brain Na + channels the same effects as described above can be achieved using phorbol esters. In our laboratory we have shown that OAG can reduce sodium currents in voltage clamped N1E-115 neuroblastoma and also inhibits sustained repetitive firing in rat cultured cortical neurons (Fig. 2AB).

Furthermore, activation of G-protein coupled receptors, including the muscarinic acetylcholine (mAChR) receptor and the dopamine (D1) receptor that are coupled to the PKC and PKA pathways, respectively, also results in reduction of Na + currents. The effects of the reduced Na + current and slowing of inactivation produced by activation of PKC are evident in pyramidal neurons of the hippocampus. Activation of mAChRs using carbachol converts intrinsically burst firing CA1 neurons into a pattern of regular spiking [43,44]. This is achieved by a reduction in not only the transient Na + current but also the persistent current crucial for determining spike threshold. Similar effects can be seen in hippocampal, striatal and cortical neurons where activation of the D1 receptor pathway reduces the number of spikes in an evoked burst.

Neuromodulation of sodium channel function, particularly somatic channels responsible for spike generation, may have an important influence upon the firing patterns of many neurons. The ability to activate PKC or PKA directly by mimicking endogenous second messengers or indirectly via GPCRs to produce reduction in both persistent and transient Na + currents may prove an exciting target for future AED design. Many large drug companies now use high- throughput combinatorial screens to highlight new leads and kinase effectors are the focus of much attention in a variety of therapeutic areas. To date, however, there are no known clinical or experimental agents that exploit such pathways to produce anticonvulsant effects.

DRUGS ACTING UPON VGSCs

A number of anticonvulsant molecules in the pharma- copoeia today have primary modes of action that involve modulation of neuronal VGSCs. The classical compound in this class was discovered by Merritt and Putnam [45] for its ability to inhibit maximal electroshock-induced seizures (MES) in rodents. Since its discovery in 1938, phenytoin [5,5-diphenylhydantoin, DPH] (Fig. 1A) and the pro-drug

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

19

Topics in Medicinal Chemistry, 2005 , Vol. 5, No. 1 19 Fig. (2) . (A) Sustained

Fig. (2). (A) Sustained repetitive firing in current clamped cultured rat cortical neurons evoked by somatic current injection. The PKC activator OAG (20m M) markedly reduces the frequency of action potentials within the burst. (B) Sodium currents from voltage clamped mouse N1E-115 neuroblastoma cells are attenuated by application of 10m M OAG for five minutes. Cells were voltage clamped at –100mV and test pulses were delivered to 0mV.

Primidone have become first line treatments for partial and generalized tonic clonic seizures due to their ability to reduce seizures without producing marked sedation [46]. Much is now known about the mechanism(s) of action of DPH and it has become clear that the biophysical characteristics of its interaction with sodium channels are central to its efficacy as an anticonvulsant.

Early evidence upon the mechanism of action of DPH was obtained in experiments using supramaximal stimulation of frog sciatic nerve. It was discovered that DPH produced no effect upon an initial action potential triggered by stimulation but blocked a second rebound spike [47]. This appeared to suggest DPH would selectively inhibit high frequency neuronal firing, a hypothesis that has been confirmed in several studies since. Notably high frequency evoked action potentials in mouse spinal cord neurons were inhibited by DPH without impairment of spontaneous low frequency action potentials [48].

Further and more extensive voltage clamp studies have delineated much about the biophysical interaction of DPH with sodium channels. Several studies using mouse neuroblastoma have shown that DPH produces tonic block of the transient sodium current I Na and that this block is highly voltage dependent [49,50]. For example DPH has been reported to have IC 50 = 120m M at –85mV, but IC 50 = 10m M at –60mV [51]. Indeed the block of sodium currents by DPH in N18 mouse neuroblastoma could be reversed by up to 95% if cells were hyperpolarized to –120mV [49]. It was predicted that because the block of sodium channels followed the voltage dependence of inactivation of I Na that DPH inhibition was related to the inactivated state. Indeed DPH does shift the voltage dependence of steady state inactivation in the hyperpolarizing direction and thus reduces the population of channels available for activation at any

given potential [49,50,52]. The binding of DPH to the fast inactivated conformation of the sodium channels is relatively slow and this, along with the strong use and voltage dependence, explains how the molecule is able to selectively inhibit high frequency epileptiform discharges whilst leaving normal cellular activity unaffected. In addition it has been reported that DPH is able to reduce I NaP in dissociated cortical neurons [53], and that this translates to a reduction in cellular excitability in layer V neurons maintained in an intact slice (it is noteworthy that DPH concentrations were lower than those required to inhibit I Na [54]). Single channel experiments revealed that DPH preferentially impairs late channel openings rather than those occurring immediately in response to a voltage step which may explain the reduction of persistent currents. A more recent study however disputes this claim showing that low micromolar concentrations of DPH produced little or no effect upon I NaP [55]. Carbamazepine [5H-Dibenz[b,f]azepine-5-caboxamide, CBZ] (Fig. 1A) is an iminostilbene derivative of tricyclic antidepressants (structurally it is completely different to DPH in that it does not contain the ureide moeity traditionally thought to be important to anticonvulsant molecules) [56]. However CBZ demonstrated shared anticonvulsant properties in vivo with DPH in potently inhibiting hindlimb extension in the MES seizure test whilst proving relatively ineffective against pentylenetetrazol (PTZ) induced epileptiform activity [57]. To complement this pre- clinical data, since its therapeutic launch CBZ has been successfully used in the control of generalised (tonic-clonic) convulsive seizures and partial (focal motor or complex partial) seizures, a niche common with DPH [58].

of

action of CBZ were derived from its ability (or 10,11-

Like

DPH

early

clues

towards

the

mechanism

20

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

epoxycarbamazepine) to inhibit, at clinically relevant concentrations, repetitive firing in cultured mammalian CNS neurons [59]. Further and more detailed voltage clamp studies in mouse neuroblastoma cells [49,52] showed CBZ produced tonic block of I Na that was highly voltage dependent; with approximately 40% of I Na blocked by CBZ (20m M) at a holding potential of –75mV, but virtually zero block when held at –120mV [49]. These studies also showed that, like DPH, the effects of CBZ were use dependent and block was accumulated with repetitive stimulation (Fig. 3).

CBZ produces shifts in the voltage dependence of steady state fast inactivation (in the hyperpolarzing direction) by stabilizing the inactivated conformation of the sodium channel and thus reduces the ability of the channel to revert the resting state (Fig. 3). The time constant for recovery from fast inactivation is also prolonged by CBZ.

Differences in the binding kinetics of DPH and CBZ to the sodium channel have been described with the latter binding 5 times faster than the former but with three fold lower affinity [60]. This may give CBZ an advantage in the treatment of seizures characterized by comparatively brief depolarizing discharges and may explain differential responses within certain patient sub groups to these agents. Although it is still commonly used in the clinic, toxicological drawbacks related to CBZ (including hepatic toxicity) have led to the development of structural congeners that maintain anticonvulsant efficacy whilst eliminating, to some extent, the toxicity. Oxcarbazepine [OXC] (Fig. 1A) shares with CBZ the dibenzazpine nucleus bearing the 5-carboxamide substituent but is structurally different at the 10,11- position. It appears that this substitution results in altered biotransformation when compared to CBZ bypassing the 10,11-epoxide metabolite thought to be responsible for the

10,11-epoxide metabolite thought to be responsible for the Fig. (3). Voltage-gated Na + channels are important

Fig. (3). Voltage-gated Na + channels are important targets for a number of clinically important anticonvulsant drugs including carbamazepine (100 m M throughout). A. Voltage clamped currents showing tonic block exerted by CBZ at two different holding potentials (test pulse applied to elicit the peak inward current at 0.5 Hz). As shown in the graph on the right, the proportion of the current blocked is increased with depolarization (note that, over the time course of these experiments, the currents had a slight tendency to increase or “run-up” due to dephosphorylation of the Na + channel complex). B. The tonic block can be enhanced by activating the channels at higher frequencies across the physiological range (up to 200Hz). The marked facilitation of block is clearly shown by progressively diminishing currents in the train (overlays are shown in inset) evoked in this experiment at 10 Hz for 3s. C. Paired pulse protocols (top right) can be used to show that CBZ generates a large hyperpolarizing shift in the steady-state inactivation profile for the Na + current and (not shown) it retards recovery from the fast-inactivated state. CBZ, LTG and DPH exert a broadly similar effect (exhibiting subtle differences in kinetics, voltage-dependence or frequency dependence). The concensus interpretation of these data are that the anticonvulsant drugs bind preferentially to (and stabilize) inactivated channel states. This preferential interaction with deploarised, ectopic and rapidly firing channels (in diseased tissue) underpins the therapeutic ratio of these clinically effective anticonvulsants. All experiments illustrated above (Errington and Lees Unpublished) were conducted on NIE115 murine neuroblastoma (in which native evoked Na + currents are tetrodotoxin sensitive) . Data are presented as Mean with SEM represented by vertical error bars (.n=3-4).

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

21

toxicity associated with the parent molecule [61]. Further modification has led to (S)-(-)-(10)-10-acetoxy-10,11- dihydro-5H-dibenz/b,f/azepine-5-carboxamide (BIA 2-093) (Fig. 1A) which was found to be particularly effective in preventing MES induced seizures with similar potency to CBZ but with fewer cognitive and motor deficits. Like CBZ and OXC, BIA 2-093 has been shown to inhibit VGSCs in a frequency and voltage dependent manner in N1E-115 cells confirming a shared mechanism of action with the former [62]. Secondary to the state dependent block of VGSCs CBZ, OXC and BIA 2-093 have all been shown to inhibit veratrine induced neurotransmitter (glutamate, aspartate, GABA, dopamine) release in rat striatal slices.

Lamotrigine [3,5-diamino-6-(2,3-dichloro-phenyl)-1,2,4- triazine , LTG] (Fig. 1A) is the first of a new generation of drugs developed in the 1980’s and 90’s to have sodium channel inhibition as its primary mechanism of action. Although between 60-80% of patients responded well to existing agents some patients did not have seizures effectively controlled. The observation that the folates possessed convulsant activity in animals led to the screening of putative antifolates as potential anticonvulsant candidates. From these screens LTG emerged as the lead compound showing similar pharmacological properties as CBZ and DPH [63-65]. Early evidence implicating the VGSC as the primary target for LTG was derived from neurochemical studies in which glutamate and GABA release evoked by veratridine (but not potassium) were suppressed in slices of rat cortex [64]. This was later confirmed by whole cell patch clamp [66] studies on cultures of rat cortical neurons in which LTG reduced the incidence of synaptic events significantly without affecting evoked postsynaptic responses. It was also demonstrated that while LTG did not attenuate single action potentials it did markedly reduce burst firing [67-68]. Voltage clamp studies in N4TG1 mouse neuroblastoma confirm that LTG exhibits similar biophysical interactions with VGSCs compared to CBZ and DPH. LTG tonically inhibits I Na in a concentration dependent manner from a holding potential of –80mV but like CBZ and DPH the block is highly voltage dependent and can be reversed by hyperpolarization. The voltage dependence of steady state inactivation is also shifted in the hyperpolarizing direction to a similar extent to CBZ and DPH [52]. It has been proposed that LTG may be unique in acting on the slow inactivated state of the channel, rather than classical fast inactivation gating, but whether this truly represents the physiological slow inactivated state or slow binding kinetics with the fast inactivated state is unclear. Like DPH, LTG has been shown to block I NaP in rat neocortical neurons, but the degree of block is less than 10% at a concentration of 10m M. Interestingly two derivatives of LTG (sipatrigine, 202W92) that are putative neuroprotective agents, are able to inhibit I NaP far more profoundly with 70 and 90% inhibition respectively at 10m M [55].

There is striking and widely accepted evidence that all (PHT, CBZ & LTG) these anticonvulsants and another group of drugs that modulate the VGSCs, the local anesthetics (LAs), do so by binding to a receptor site inside the cytoplasmic vestibule of the ion conducting pore of the channel. More specifically if either F1764 or Y1771 are mutated to alanine in transmembrane segment IVS6 the

resultant sodium channels are far less susceptible to voltage and frequency dependent block by LAs [69,70]. The similarity between the blocking action of the LAs and the anti-epileptic drugs (AEDs) suggested that this binding site may be shared across both classes of molecules and indeed the F1764A and Y1771A mutants showed greatly attenuated block by DPH and LTG [11]. Furthermore Kuo [56] has shown that applications of mixtures of LTG, DPH and CBZ cannot produce double occupancy of the channel. He concludes that despite dissimilarity in structure, these drugs (and probably CBZ derivatives OXC and BIA 2-093) likely share the same or very similar receptor site on the sodium channel a subunit.

Despite the effectiveness of the aforementioned AEDs in controlling seizures, in patients with generalized convulsive seizures, some patient groups are still found to be refractory to existing therapies. This has led to further development of anticonvulsant compounds despite negative market forces including long lead times and brief patent life of new molecules. Some of these newer drugs, discovered by screening large libraries of compounds in various animal models, have been shown to act (to some degree) on VGSCs. Topiramate [2,3:4,5-bis-O-(1-methyl-ethylidene)-36-D- fructopyranose sulfamate, TPM] (Fig. 1A) is an anticonvulsant drug which was licensed for clinical use in many European countries in the late 1990’s. In the traditional animal models (MES, PTZ) TPM showed a profile similar to that of DPH [71] and it has therefore become clinically established as an add-on therapy in simple or complex partial seizures with or without secondary generalization. The mechanism of action of TPM, although still unclear appears to exhibit three facets, namely: blockade of kainate evoked excitatory currents [72], enhancement of inhibition through GABAergic chloride channels [73] and depression of sodium currents [74]. TPM significantly reduced sustained repetitive firing in cultured hippocampal pyramidal cells at therapeutically relevant (10-30m M) concentrations and this was later confirmed to be due to attenuation of I Na . In cerebellar granule cells [75] and dissociated neorcortical neurons [76], TPM produced voltage dependent tonic block of I Na and shifted the steady state inactivation curve to more negative values in a fashion previously described for LTG, CBZ, DPH, OXC and BIA 2-093. TPM has also been reported to inhibit sustained sodium conductances (I NaP ) in evoked in neocortical neurons in response to slowly depolarizing membrane potential ramps at concentrations lower than those required to inhibit I Na . In this way TPM mimics DPH and, furthermore, it is conceivable that the inhibition of the persistent current may represent a major contribution to the reduction in cellular excitability produced by all these drugs. The tetronic acid derivative Losigamone [(±)-5(R,S)-5-

(2-chlorophenyl)hydroxymethyl)-4–methoxy(5H0-furanone,

LSG] (Fig. 1A) is an experimental anticonvulsant currently undergoing phase II/III clinical trials in patients with partial and secondary generalised seizures. LSG has been shown to produce a reduction in spontaneous synaptic activity in cultured hippocampal neurons and a marked suppression of sustained repetitive firing evoked by somatic current injection [77]. However the effects of LSG are apparently different from many classical sodium channel-affecting

22

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

anticonvulsants previously described. In the majority of cellular studies inhibition of SRF can be closely correlated to impairment of I Na . In whole cell recordings made from hippocampal neurons in brain slice preparations LSG did not inhibit I Na at relevant concentrations. LSG did however produce inhibition of I NaP and it has been suggested that this may prove to be crucial to its ability to decrease neuronal hyperexcitability [78]. In this respect LSG may represent the first compound to be used in epilepsy that inhibits I NaP without significant effect on transient sodium currents. Another new anticonvulsant ligand which acts upon voltage gated sodium channels is currently in phase III trials. Safinamide [Propanamide, 2-[[[4-[(3-fluorophenyl)methoxy] phenyl]methyl]amino]-, (2S)-monomethanesulfonate, PNU- 151774E, NW-1015, SAF] (Fig. 1A) produced inhibition of sustained repetitive firing, inhibition of TTX sensitive sodium currents and displayed higher affinity for the Na + channel [ 3 H] batrachotoxin receptor site (site 2) than phenyoin or lamotrigine (EC 50 : 8.2m M) [79]. Although the actions of SAF appear to be complex and multifaceted, including inhibition of L and N-type calcium channels, the compound nonetheless represents another molecule whose anticonvulsant actions are conferred by interaction with VGSCs. Clearly the VGSC represents a major ion channel target for both existing clinical and experimental anticonvulsant molecules. It is apparent that despite varied chemical structures many compounds can interact to produce state dependent block of I Na and that in some cases there is overlapping reduction of I NaP providing a dual mechanism for reducing hyperexcitability.

STRUCTURE, FUNCTION AND ANTICONVULSANT PHARMACOLOGY OF VOLTAGE GATED CALCIUM CHANNELS The voltage gated calcium channels (VGCCs) are critical for the proper functioning of excitable cells within the

central nervous system. Calcium influx into the cellular interior occurring in response to channel gating is essential for the translation of transient electrical signals into biochemical events, both acutely, as in neurotransmitter release, and more chronically, as in regulating gene expression and even cell-death. VGCCs were initially described by Paul Fatt and Bernard Katz in crustacean muscle but have subsequently been shown to exist in an array of mammalian cells types including, crucially, CNS neurons. VGCCs were first classified according to their pharmacological and biophysical characteristic into P/Q, N, L, R (high voltage activated, HVA) and T (low voltage activated, LVA) types [80]. HVA channels have been extensively characterised biochemically revealing that VGCCs are complex heteromeric structures. VGCCs have been demonstrated to be formed by at least three main subunits namely a 1 , a 2 d and b, as well as, in some cases a g subunit. The main component of the VGCC is the approximately 2000 amino acid 190-250kDa a subunit. The VGCC a subunit is structurally similar to VGSC a subunits, with four repeated domains each being produced by six transmembrane spanning segments. As with its sodium and potassium conducting counterparts it is predicted that the calcium channel voltage sensor is the S4 membrane spanning segment and the pore is formed by a re-entrant loop between the S5 and S6 segments (Fig. 4B). Ten VGCC a subunits have thus far been identified through homology screening and this has led to reclassification of the different Ca channel types. The cloned subunits are know divided into three functionally related families based upon their biophysical and pharmacological characteristics [81]. Ca v 1.1-1.4 subunits form the L-type channels whilst Ca v 2.1, Ca v 2.2 and Ca v 2.3 form the P/Q, N and R types respectively. The LVA T-type channels include an a subunit from the Ca v 3.1-3.3 types [80].

2+

The first of the calcium currents to be described was the HVA L-type (because the current is long lasting, particularly

L-type (because the current is long lasting, particularly Fig. (4) . (A) Structures of anticonvulsant drugs

Fig. (4). (A) Structures of anticonvulsant drugs acting at VGCCs. (B) Diagram of the subunit structure of the VGCC a subunit and the accessory b , a 2 and d subunits.

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

23

when Ba 2+ is used as the permeant ion [82]) current in smooth and skeletal muscle. The L-type channels are characterized by slow voltage dependent inactivation, high voltage of activation, modulation by cAMP dependent phosphorylation pathways and inhibition by dihydro- pyridines [83]. The T-type LVA calcium channels that were discovered in cerebellar purkinje neurons [84] but characterised in detail in dorsal root ganglion neurons [82]. The T-type designation is based upon the rapid inactivation of this type of channel resulting in transient currents. Furthermore the T-type channels are activated at much more negative membrane potentials, have small single channel conductances and are insensitive to block by dihydropyridines. A further type of Ca 2+ current was revealed based upon kinetic and pharmacological divergence from the existing L and T-type currents. Nowycky et al. showed using whole cell and single-channel recording from dorsal root ganglion neurons a calcium current that activated at more negative potentials than did the L-type but more positive than the T- type [82]. The new current, which became known as the N- type, also showed more rapid inactivation than L but slower inactivation than the transient type. However it is the sensitivity to block by the snail peptide w -conotoxin GVIA that is the crucial distinguishing factor for identification of N-type channels [85]. The remaining P, Q and R-type calcium channels were identified in a range of neuronal types based upon sensitivity to other peptide toxins. P-type currents are highly sensitive to block by the spider toxin w - agatoxin IVA and were first recorded in purkinje neurons whilst the Q-type were first found in cerebellar granule neurons [86] and display much lower affinity for the toxin. Finally the R-type were found also in cerebellar granule neurons and take their name from the fact they are resistant to the subunit specific organic and peptide Ca 2+ channel blockers [86]. As described above, VGCCs are formed by a pore forming a 1 subunit linked with several accessory subunits. As with the VGSC these accessory subunits are not required for the formation of functional ion conducting pores but are essential for conferring correct gating kinetics and cell surface expression of the ion channel. The VGCC b subunits (b 1-4) are 53-65kDa proteins found within the intracellular compartment which induce large changes to the voltage dependence of activation and the rate of inactivation. VGCCs also include an a 2 d (a 2 d 1-4) subunit which are 123- 129kDa dimers that are linked to the a 1 subunit by a dispulphide link. The a 2 component is a membrane spanning protein whilst the d component is found extracellularly (Fig. 4B). The a 2 d subunit is also thought to modulate channel gating kinetics and regulate levels of expression but to a lesser degree than the b subunit [80]. Furthermore several 25-36kDa g (g1-5) subunits have been identified but the expression and functional status of this subunit is still relatively unknown.

There is considerable evidence available (reviewed recently by Jones [87]) concerning the role of VGCC in epileptogenesis although there are few available AEDs that target the VGCC to exert their anticonvulsant effects. In particular genetic mutations in human calcium channel genes (table 1) or those of several mouse species have been

associated with epileptic phenotypes. For example the stargazer mouse has shown that mutations of g subunits result in loss of g 2 subunits without compensatory up regulation of other g subunit types. It has been suggested by some authors that this may contribute to the pathogenesis in this mutant mouse strain that results in spike wave seizures characteristic of absence epilepsy [87]. This is still somewhat controversial however as some groups implicate the mutant g subunit analogue (stargazin protein) an important role in molecular trafficking and cell surface expression of AMPA receptors [88].

The LVA T-type VGCC has been implicated in playing a significant role in controlling rhythmic discharges in thalamocortical neurons that display epileptiform spike wave discharges associated with absence seizures. 2-Ethyl-2- methylsuccinimide [Ethosuximide, ESM] (Fig. 4A) is an AED that has an unusual clinical profile. ESM shows efficacy against absence seizures but not against partial or generalized tonic-clonic seizures. This has been shown to be due to the ability of ESM to partially inhibit T-type calcium channels at therapeutically relevant concentrations and hence reduce the hyperexcitability of thalamocortical neurons that is associated specifically with absence seizures [89]. Furthermore it has also been demonstrated that methyl- phenyl suximide (the active metabolite of the related compound methsuximide) but not its inactive analog also produces inhibition of T-type currents in thalamic neurons [90]. To complement these findings a recent study has shown that both of these agents are able to inhibit cloned human T- type VGCCs expressed in HEK293 cells whereas non- anticonvulsant congeners were ineffective [91].

A recent and interesting development in the field of

VGCC anticonvulsant pharmacology are the novel drugs 1- (aminomethyl) cyclohexanacetic acid [Gabapentin, GBP] and S(+)-3-isobutyl-gamma-amniobutyric acid (Pregabalin, PGL) (Fig. 4A). GBP has proven to be effective against a wide range of seizure models in animals and clinically useful for the treatment of partial seizures. It has been approved as an add-on therapy in the U.K. since 1992 and the U.S. since 1993. GBP was designed to be a CNS penetrating GABA analog but was later found to have no effect at the GABA receptors, enzymes or transporters.

A putative binding site for GBP was first identified on

the a 2 d subunit of HVA VGCCs using [ 3 H]-gabapentin on purified porcine brain [92]. The a 2 and d components were found to both be required for binding of GBP although the disulphide link between the two did not necessarily need to be intact. Binding of GBP to these subunits also appears to be subunit specific with higher affinity binding to a 2 d -1 than a 2 d -2 and no binding at a 2 d -3 and a 2 d -4. This binding

evidence is supported by electrophysiological data in a number of different preparations that show inhibition of calcium currents. Several studies report inhibition of HVA calcium channels in dissociated rat brain neurons and dorsal root ganglion cells [93,94], reduction of Ca 2+ influx into synaptosomes through P/Q-type channels and decreased glutamate release [95]. Recent experiments by Bayer and colleagues have shown that GBT selectively blocks P/Q-type

Ca

suggest that this may result from interaction of the GBP binding a 2 d -1 or a 2 d -2 subunits with the a 1A pore forming

2+

channels in dorsal horn neurons (Fig. 5). The authors

24

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

in Medicinal Chemistry, 2005 , Vol. 5, No. 1 Lees et al. Fig. (5) . (A)

Fig. (5). (A) Postsynaptic responses to exogenously applied glutamate (1mM) and glycine (1mM) to whole cell patch clamped dorsal root neurons. Gabapentin (1m M) does not significantly effect the amplitude or duration of either the evoked excitatory or inhibitory currents. (B) AMPA-EPSCs and GLY-IPSCs evoked by extracellular stimulation are significantly attenuated by gabapentin (1m M) when N-type channels are blocked but not when P/Q-type channels are blocked. This demonstrates gabapentin is able to preferentially block P/Q-type calcium channels in sensory neurons (from ref. [95]).

subunit of the P/Q-type channels [96]. Although other potential mechanisms have been proposed for GBP including enhancement of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), it is now generally accepted that actions of GBP and that of its follow-up compound PGL are at least in part mediated by interaction with a 2 d- 1/2 subunits of VGCCs. The clinically important AEDs LTG and TPM have also shown some activity against VGCCs although modulation of VGSC is widely accepted as their primary mode of action. Patch clamp experiments have shown that TPM is able to produce inhibition of L-type VGCCs in dentate granule cells [97] whilst in rat cortical neurons LTG produces concentration dependent reduction in N and P but not L-type currents [98].

STRUCTURE, FUNCTION AND ANTICONVULSANT PHARMACOLOGY OF VOLTAGE GATED POTASSIUM CHANNELS

Potassium channels that are gated by changes in membrane potential represent a functionally and structurally diverse family of ion channels that are crucial for many key cellular processes including setting resting membrane potential, determining firing threshold, action potential repolarization [14] and determination of postsynaptic excitability. They represent one of the greatest unexploited targets for future AED development: only a few experimental compounds evidently target these channels as their primary mode of action. Several families of mammalian K + channels exist, including those gated by a change in membrane potential (K v ); those which favor the movement

of K + ions into the cell rather than outward (K ir ); and the recently discovered twin pore K channels (KCNK, TWIK, TRAAK and related channels) that appear to generate neuronal resting membrane potential. The largest of these “superfamilies”, and also the first to be discovered, are the channels that give rise to I Kv (and I KA ) currents. The K v channel sub-units are the best characterised of the K + channels in terms of both structure and function. The first of the K v channels to be cloned were those from the Drosophila fruit fly voltage gated K + channel genes Shaker, Shal, Shab and Shaw but since, a further 29 related members of the K v family have been discovered. These have been divided into eight gene families each having several members {KCNA (K v 1.1-1.8); KCNB (K v 2.1-2.3); KCNC (K v 3.1-3.4); KCND (K v 4.1-4.3); KCNF (K v 5.1); KCNG (K v 6.1-6.4); KCNS (K v 9.1-9.3); KCNV (K v 8.1-8.3)} and can form homo or heteromeric channels (K v 1, K v 2, K v 3 and K v 4) with other subunits within their own family.

As previously discussed, K v channels, are typically formed by tetrameric assemblies of K v a- subunits [99] each of which having six transmembrane spanning domains (S1- S6) much like a single sodium channel repeated motif (Fig. 6C). The S4 transmembrane spanning segment provides the voltage sensing capacity [100] whilst the pore of the complete channel is formed by the re-entrant loop between S5-S6 (P-loop). The P-loop contains the structural motif GYG which forms the basis of the selectivity filter [101] and makes the channel selective for K + ion conductance. MacKinnon’s group has made a huge contribution to our knowledge of the fine molecular structure of voltage gated

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

25

Topics in Medicinal Chemistry, 2005 , Vol. 5, No. 1 25 Fig. (6) . (A) Structures

Fig. (6). (A) Structures of anticonvulsant drugs acting at VGKCs. (B) Diagram showing the structure of potassium channel a subunits including the voltage gated (K v ), KCNQ and the twin pore KCNK type channels. (C) Differences in subunit composition of complete ion channels. Na + and Ca 2+ channels are homomers comprising a single a subunit whilst potassium channels are formed by heteromeric assembly of four smaller a subunits.

K+ channels through X-ray crystallographic studies of related bacterial channels [102]

Due to the diversity of K + channels brought about by many gene families, splice variation and heteromeric assembly (see subunit structures in Fig 6C) the range of physiological properties and roles of these channels are extremely diverse. The K v channels can however be classified into three broad groups based upon their voltage dependence of activation and the extent to which they inactivate. At resting membrane potential K v channels reside in a closed state and are activated in response to depolarization whereupon they allow K + ions to flow from the cellular interior resulting in hyperpolarization of membrane potential. The first functional class of K v channels are the so-called low voltage activated channels (LVA, e.g. Kv1.1, KCNA). These are non-inactivating channels that produce sustained outward potassium currents in response to relatively low changes in membrane potential away from rest (~ -60mV). LVA channels activate and deactivate slowly and as such are often known as delayed rectifier channels. The slow kinetic features associated with LVA channels means they are important in determining spike frequency adaptation but have little effect upon single spikes or the duration or rise time of the first spike within a burst. The second major subtype of the delayed rectifier family, are the high voltage activated channels (HVA, e.g. K v 3, KCNC). HVA activate and deactivate more rapidly than LVA channels but activation only occurs in response to large depolarization, such as that brought about by an action potential. HVA channels are therefore mostly responsible for the repolariz- ation phase of the action potential and allowing high frequency neuronal firing. This is typified by expression of

K v 3 channels in cortical interneurons where it has been correlated to the short duration action potentials that allow very high firing frequencies. The significant role the delayed rectifier channels play in controlling cellular excitability and burst firing frequency makes them an ideal therapeutic target that may be exploited for anticonvulsant benefit.

Levetiracetam [(S)-a -ethyl-2-oxo-pyrrolidine acetamide, LEV] (Fig 6A) is a pyrrolidine derivative that is a well-

tolerated adjunctive therapy for refractory partial seizures in adults. Despite its clinical success the mechanism of action of LEV has remained elusive with GABAergic facilitation,

inhibition of Na + /low voltage activated Ca

inhibition of excitatory ionotropic conductances being ruled out as potential candidates [103,104]. Complementing this lack of activity at established molecular target sites for anticonvulsants in vitro, is the notable observation that LEV differs from existing anticonvulsants in showing lack of activity against MES and PTZ induced seizures in vivo. Recently however Madeja and colleagues [105] have postulated that the anticonvulsant properties of LEV may be substantially contributed to by a reduction in the conductance of delayed rectifier K + channels. The authors of this study demonstrated using acutely isolated rat and guinea pig CA1 neurones, and the whole cell patch clamp technique, that a 26% reduction in the delayed rectifier K + current is produced by 100m M LEV. This inhibition was demonstrated to be sufficient to produce marked impairment of repetitive action potential firing. This study implicates LEV as the first compound to have its primary mode of action as a blocker of delayed rectifier channels although no distinction is made between the LVA and HVA types.

2+

current and

26

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

The final major functional type of K v channels are those channels which are capable of inactivation and give rise to the so called transient outward K + current or A currents. These channels activate more rapidly than the delayed rectifier type but also rapidly and spontaneously deactivate owing to a ‘ball and chain’ or slower C-type inactivation mechanism depending upon the subunit composition of the channel. There is no evidence to date that any existing clinical or experimental drugs exploit modulation of A- currents as their primary mode of action. However it has been reported that LTG and CBZ are able to potentiate outward K A conductance at concentrations similar to those at which they exert sodium channel inhibition [106,107]. 4-AP models owing to the occlusion of a primary target by the convulsant toxin.

The M-current (I M ) is a slowly activating, non- inactivating and slowly deactivating current that was first described in frog sympathetic ganglia by Brown and Adams in 1980 [108]. The name is derived from the ability of muscarinic receptor stimulation to inhibit the K+ current and this has been shown to be present in a number of CNS

neurons [109]. I M has an important role in determining neuronal excitability as it represents the only sustained inhibitory current in the range of potentials around spike threshold. The slow activation and deactivation kinetics of I M mean that it plays a significant part in controlling resting membrane potential, responsiveness to synaptic input and as

a braking mechanism on repetitive firing. The molecular

identity of the M-current was unknown until it was discovered that currents induced by heteromers of KCNQ2/KCNQ3 potassium channel subunits shared similar pharmacological and biophysical properties to native I M [110]. The KCNQ genes encode a growing family of K + channel products that are structurally related to the K v channel a subunits and like the classical K v channels are gated by changes in membrane potential. They have six transmembrane spanning domains, a single pore forming loop that forms the selectivity filter and sequences of charged amino acids in the S4 domain that probably represent the voltage sensor (Fig. 6BC). Although it has not been shown directly, KCNQ subunits, of which there are five known types (KCNQ1, KCNQ2, KCNQ3, KCNQ4 and

KCNQ5), likely form homo or heterotetramers to produce functional channels [111]. The significance of these channels

in the control of cellular excitability is demonstrated by the

observation that mutations of the KCNQ 2/3 channels result

in the rare condition of benign familial neonatal convulsions

(BFNC, table 1) [112,113]. BNFC mutations in either KNCQ2 or KNCQ3 may only produce a reduction of between 20-30% [114] in the current amplitude of the expressed heteromeric channels but this is often sufficient to induce tonic-clonic seizures in the neonate.

Owing to the clear link between, an albeit rare idiopathic form of epilepsy, KCNQ2/3 mutations and the correlation between these channel subunits and the M-current (that is so crucial for determining cellular excitability), it would appear that K + channel opening represents an ideal therapeutic target for novel anticonvulsant ligands. Indeed an experimental anticonvulsant ligand already undergoing Phase II clinical trials has been shown to act, at least in part, through opening KCNQ2/KCNQ3 potassium channels.

Retigabine [(D-23129, N-(2-amino-4-(4-fluorobenzylamino)- phenyl) carbamic acid ethyl ester, RTG] (Fig 6A) is structurally unrelated to any currently marketed anticon- vulsant drug and has been shown to exert anticonvulsant effects in broad range of seizure models. It was first found that RTG activated a K + conductance in marginally depolarized NG108-15 neuronal cells at concentrations approximating to therapeutic plasma levels [115]. Although it was unknown at the time of this study it was later observed that the pharmacological profile of the RTG induced current was consistent with that of the M-current. It has since been confirmed in Chinese hamster ovary (CHO) cells transfected with either KCNQ2 (homomer) or KCNQ2/KCNQ3 (heteromer) that RTG is indeed that first molecule in a new class of KCNQ K + channel opening drugs [116] (Fig. 7). The direct interaction of Retigabine with the KCNQ channels that are responsible for BFNC may be able to overcome the small loss of function underlying this rare epileptic condition and illustrates the importance of molecular biology and genetics to the development of therapeutic agents for the treatment of idiopathic syndromes.

A further important family of potassium channels has been recognized only within the last eight to ten years. Leak currents (resting or background conductances) have been described in physiology for many years but only in 1996 was it discovered that these currents were carried by discrete molecular entities within the cell membrane. The first of these ion channel structures was cloned from the neuromuscular junction of Drosophila and became known as KCNK0. Later in the same year the first mammalian gene for this type of channel subunit, KCNK1, was also discovered although this was found to be physiologically non- functional. Although the differences in structural and functional properties associated with KCNK channels are too extensive to cover fully in this review (reviewed in detail by Goldstein et al. [117]) there is some interesting pharma- cology related to these channels that deserves attention. Briefly the KCNK family of K+ channels subunits differ in that they have two pore forming or P domains and four (rather than six) transmembrane spanning segments (Fig. 6B). They are open at rest and are subject to modulation by a number of physiologically pertinent agents including cyclic nucleotides, noradrenaline, serotonin and fatty acids. This type of ligand modulation at KCNK channels is capable of influencing channel gating and conductance properties and as such is predicted to contribute to control of membrane potential and cellular excitability.

The neuroprotective agent sipatrigine (STG), which is chemically related to LTG, has been shown to potently inhibit at least two of the two pore domain channels (that are known to be sensitive to arachidonic acid), namely KCNK2 (also known as TREK-1) and KCNK4 (also known as TRAAK) when they are expressed in HEK293 cells [118]. If one assumes that the KCNK channels are the main targets for the neuroprotective actions of STG it may be expected that inhibition of the leak conductance would result in depolarization and increased excitability/excitotoxicity. However, if KCNK2/4 were preferentially expressed on GABAergic interneurones, reduced net excitability may be produced by an increase in polysynaptic inhibitory tone. Alternatively chronic depolarization resulting from the

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry,

2005, Vol. 5, No. 1

27

Topics in Medicinal Chemistry, 2005 , Vol. 5, No. 1 27 Fig. (7) . The anticonvulsant

Fig. (7). The anticonvulsant agent retigabine is mechanistically novel. It produces (A) a concentration dependent increase in potassium conductance through KNCQ2/KNCQ3 heteromers expressed in CHO cells by directly opening the channel. (B) Retigabine also shifts the voltage dependence of activation to more negative potentials in a reversible fashion. This results in the cellular potential being taken further from the threshold potential for spike firing (from ref. [115]).

decreased leak conductance may cause reduction in excitatory neurotransmitter release by depolarization mediated block of glutamatergic neurons. Although the anticonvulsant compound LTG produces only <10% block of this current at 10m M [118] it is important to consider the other mechanisms by which the anticonvulsant is known to act. A small reduction in the KCNK2/4 current produced by LTG application may, on its own, produce only insignificant changes in excitability but the resulting depolarization would undoubtedly recruit more inactivation of both sodium channel and calcium channels. It is known that LTG binds potently to the inactivated conformation of the sodium channel (as above) to reduce electrogenesis and also reduces excitatory neurotransmitter release that likely results from reduction in availability of calcium channels in the presynaptic terminal. In these terms, what looks like a paradoxical excitant or pro-convulsant surrogate effect of the drug may represent a synergistic pathway to promote its potency in blocking epileptiform activity. This type of structure-dependent, non-selective cross talk with other channels may explain the diverse pharmacological profiles of DPH, CBZ and LTG in the living brain (despite their commonality at the VGSCs).

Clearly the functional diversity amongst the K + channels make them prospective targets for future AED development. The production of selective modulators of the various K + channel types may serve to further relieve the symptoms of those patients with refractory or idiopathic epilepsies whose conditions are poorly controlled by the existing compounds.

CONCLUSION

To sum up we wish to stress that voltage-gated ion channels have been crucial in the treatment of epilepsy (often their mode of action emerged after serendipitous discovery of their clinical promise). One by one the large multi- national pharmaceutical companies are withdrawing from biorational drug discovery in the epilepsy field (because of the prohibitive development costs and the long lead times). This is clearly detrimental to the significant number of

refractory patients: there is a surprisingly large incidence of sudden death in epilepsy patients (SUDEP) and a clear clinical need for new drugs. The market analysts appear to be ignoring completely the obvious promise that such drugs hold in the massive pharmaceutical market of neuropathic pain. These chronic pain syndromes are notoriously difficult to treat but licensed anticonvulsants (even those with overt side effects in the form of sedation, impairment of hepatic function and serious blood dyscrasias are often useful in the treatment of chronic pain). Gabapentin has become the gold- standard in the treatment of conditions like post-herpetic and trigeminal neuralgia. The NIH in the USA continue to fund drug discovery research in epilepsy and it seems likely that novel drugs in the future will exploit the superfamilies of ion-selective pores reviewed here.

REFERENCES

[1]

Traub, R. D.; Borck, C.; Colling, S. B.; Jefferys, J. G. On the

441-456.

[2]

structure of ictal events in vitro. Epilepsia 1996, 37, 879-891. Jefferys, J. G. R. Models and Mechanisms of Experimental

[3]

Epilepsies. Epilepsia 2003, 44, 44-50. Hodgkin, A. L.; Huxley, A. F. The dual effect of membrane

[4]

potential on sodium conductance in the giant axon of Loligo. J. Physiol. (London) 1952, 116, 497-506. Hodgkin, A. L.; Huxley, A. F. The components of membrane

[5]

conductance in the giant axon of Loligo. J. Physiol (London) 1952, 116, 473-496. Hodgkin, A. L.; Huxley, A. F. Currents carried by sodium and

[6]

potassium ions through the membrane of the giant axon of Loligo. J. Phsiol. (London) 1952, 116, 473-506. Catterall, W. A. Cellular and molecular biology of voltage-gated

[7]

Na + channels. Physiol. Rev. 1992, 72, S15-S48. Catterall, W. A. Molecular properties of brain sodium channels: an

important target for anticonvulsant drugs. Adv. Neurol. 1999, 79,

[8]

Qu, Y.; Curtis, R.; Lawson, D.; Gilbride, K.; Ge, P.; DiStefano, P.

[9]

S.; Silos-Santiago, I.; Catterall, W. A.; Scheuer, T. Differential modulation of sodium channel gating and persistant sodium currents by the b 1, b 2 and b 3 subunits. Mol. Cell. Neurosci. 2001, 18, 570-580. Goldin, A. L.; Barchi, R. L.; Caldwell, J. H.; Hofmann, F.; Howe, J. R.; Hunter, J. C.; Kallen, R. G.; Mandel, G.; Meisler, M. H.; Netter, Y. B.; Noda, M.; Tamkun, M. M.; Waxman, S. G.; Wood, J. N.; Catterall, W. A. Nomenclature of voltage-gated sodium channels. Neuron 2000, 28, 365-368.

28

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

[10]

Noda, M.; Ikeda, T.; Kayano, T.; Suzuki, H.; Takeshima, M.; Kurasaki, H.; Takahashi, S.; Numa, S. Existence of distinct sodium

[34]

Brown, A. M.; Schwindt, P. C.; Crill, W. E. Different voltage dependence of transient and persistant Na + currents is compatible

[11]

channel messenger RNAs in rat brain. Nature 1986, 320, 121-127. Ragsdale, D. S.; Avoli, M. Sodium channels as molecular targets

with modal gating hypothesis for sodium channels. J. Neurophysiol. 1994, 71, 2562-2565.

for antiepileptic drugs. Brain Res. Rev. 1998, 26, 16-28.

[35]

Huguenard, J. R.; Hamill, O. P.; Prince, A. Developmental changes

[12]

Catterall, W. A. A 3D view of sodium channels. Nature 2001, 409,

in Na + conductance in rat cortical neurons: appearance of a slowly

 

988-991.

inactivating component. J. Neurophysiol. 1988, 59, 778-795.

[13]

Hille, B. Ion channels of excitable membranes; Sinauer Assoc.:

[36]

Crill, W. E. Persistant sodium current in mammalian central

[14]

Sunderland, MA., 1992. Hodgkin, A. L.; Huxley, A. F. A quantative description of

[37]

nervous system. Annu. Rev. Physiol. 1996, 58, 349-362. Stafstrom, C. E.; Schwindt, P. C.; Chubb, M. C.; Crill, W. E.

[15]

membrane current and its application to conduction and excitation in nerve. J Physiol (Lond) 1952, 117, 500-544. Armstrong, C. M.; Bezanilla, F. Charge movement associated with

Properties of persistant sodium conductance and calcium conductance of layer V neurons from cat sensorimotor coretx in vitro. J. Neurophysiol. 1985, 53, 152-170.

the opening and closing of the activation gates of Na channels. J.

[38]

Stafstrom, C. E.; Schwindt, P. C.; Crill, W. E. Negative slope

[16]

Gen. Physiol. 1974, 63, 533-552. Armstrong, C. M.; Bezanilla, F. Currents related to the movement

conductance due to a persistant subthreshold sodium current in cat neocortical neurons in vitro. Brain Res. 1982, 236, 221-226.

of the gating particles of the sodium channels. Nature 1973, 242,

[39]

Cantrell, A. R.; Catterall, W. A. Neuromodulation of Na + channels:

 

459-461.

an unexpected form of cellular plasticity. Nat. Rev. Neurosci. 2001,

[17]

Stümer, W.; Conti, F.; Suzuki, X.; Wang, M.; Noda, M.; Yahadi,

 

2, 397-407.

N.;

Kubo, H.; Numa, S. Structural parts involved in activation and

[40]

Murphy, B. J.; Rossie, S.; DeJongh, K. S.; Catterall, W. A.

[18]

inactivation of the voltage gated sodium channel. Nature 1989, 339, 597-603. Patton, D. E.; West, J. W.; Catterall, W. A.; Goldin, A. L. Amino

Identification of sites of selective phosphorylation and dephosphorylation of the rat brain sodium channel a subunit by cAMP dependent protein kinase and phosphoprotein phosphatases.

acid residues required for fast Na+ channel inactivation: charge

J.

Biol. Chem. 1993, 268, 27355-27362.

neutralizations and deletions in the III-IV linker. Proc. Nat. Acad.

[41]

Smith, R. D. Goldin, A. L. Phosphorylation of brain sodium

Sci. U. S. A. 1992, 89, 10905-10909.

channels in the I-II linker modulates function in Xenopus oocytes.

[19]

Vassilev, P. M.; Scheuer, T.; Catterall, W. A. Identification of an

J.

Neurosci. 1996, 16, 1965-1974.

intracellular peptide segment involved in sodium channel

[42]

Li, M.; West, J. W.; Lai, Y.; Scheuer, T.; Catterall, W. A.

[20]

inactivation. Science 1988, 241, 1658-1661. West, J. W.; Patton, D. E.; Scheuer, T.; Wang, Y.; Goldin, A. L.;

Functional modulation of brain sodium channels by cAMP- dependent phosphorylation. Neuron 1992, 8, 1151-1159.

Catterall, W. A. A cluster of hydrophobic amino acid residues required for fast Na + channel inactivation. Proc. Nat. Acad. Sci.

[43]

Alroy, G.; Su, H.; Yaari, Y. Protein kinase C mediates muscarinic block of intrinsic bursting in rat hippocampal neurons. J. Physiol.

U.

S. A. 1992, 89, 10910-10914.

1999, 518, 71-79.

[21]

McPhee, J. C.; Ragsdale, D. S.; Scheuer, T.; Catterall, W. A. A critical role for transmembrane segment IVS6 of the sodium channel a subunit in fast inactivation. J. Biol. Chem. 1995, 270,

[44]

Azouz, R.; Jensen, M. S.; Yaari, Y. Muscarinic modulation of of intrinsic burst firingi in rat hippocampal neurons. Eur. J. Neurosci. 1994, 6, 961-966.

12025-12034.

[45]

Merritt, H. H.; Putnam, T. J. Sodium diphenylhydantoinate in the

[22]

Goldin, A. L. Mechanisms of sodium channel inactivation. Curr.

treatment of convulsive disorders. J. Am. Med. Assoc. 1938, 111,

Opin. Neurobiol. 2003, 13, 284-290.

1068-1073.

[23]

Traub, R. D.; Jefferys, G. R.; Whittington, M. A. Fast Oscillations

[46]

Brodie, M. J.; Dichter, M. A. Antiepileptic drugs. N. Engl. J. Med.

523-530.

[24]

in Cortical Circuits.; The MIT Press: Cambridge, MA, 1999. Featherstone, I. A.; Richmond, J. E.; Ruben, P. C. Interaction

[47]

1996, 334, 168-175. Toman, J. E. P. The neuropharmacology of antiepileptics.

between fast and slow inactivation in Skm1 sodium channels. Biophys. J. 1996, 71, 3098-3109.

[48]

Electroencephalogr. Clin. Neurophysiol. 1949, 1, 33-44. McLean, M. J.; McDonald, R. L. Multiple actions of phenytoin on

[25]

Richmond, J. E.; Featherstone, I. A.; Hartmann, H. A.; Ruben, P.

mouse spinal cord neurons in cell culture. J. Pharmacol. Exp. Ther.

C.

Slow inactivation in human cardiac sodium channels. Biophys.

1986, 237, 779-789.

J.

1998, 74, 2945-2952.

[49]

Willow, M.; Gonoi, T.; Catterall, W. A. Voltage clamp analysis of

[26]

Fleidervish, I. A.; Friedman, A.; Gutnick, M. J. Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea pig neocortical neurones in slices. J. Physiol. 1996, 493, 83-

the inhibitory actions of diphenylhydantoin and carbamazepine on voltage-sensitive sodium channels in neuroblastoma cells. Mol. Pharmacol. 1985, 27, 549-558.

97.

[50]

Matsuki, N.; Quandt, F. N.; Ten Eick, R. E.; Yeh, J. Z.

[27]

Toib, A.; Lyakhov, V.; Marom, S. Interaction between duration of

Characterization of the block of sodium channels by phenytoin in

activity and time course of recovery from slow inactivation in mammalian brain Na + channels. J. Neurosci. 1998, 18, 1893-1903.

mouse neuroblastoma cells. J. Pharmacol. Exp. Ther. 1984, 228,

[28]

Avoli, M.; Barbarosie, M.; Lucke, A.; Nagao, T.; Lopantsev, V.;

[51]

Taylor, C. P.; Meldrum, B. S. Na+ channels as targets for

Kohling, R. Synchronous GABA-mediated potentials and epileptiform discharges in the rat limbic system in vitro. J.

[52]

neuroprotective drugs. Trends Pharmacol. Sci. 1995, 16, 309-315. Lang, D. G.; Wang, C. M.; Cooper, B. R. Lamotrigine, Phenytoin

[29]

Neurosci. 1996, 16, 3912-3924. Lopantsev, V.; Avoli, M. Participation of GABAA-mediated

and Carbamazepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells. J. Pharmacol. Exp. Ther.

inhibition in ictal-like discharges in the rat entorhinal cortex. J. Neurophysiol. 1998, 79, 352-360.

[53]

1993, 266, 829-834. Chao, T. I.; Alzheimer, C. Effects of phenytoin on the persistent

[30]

Avoli, M.; D'Antuono, M.; Louvel, J.; Kohling, R.; Biagini, G.;

Na+ current of mammalian CNS neurones. Neuroreport 1995, 6,

Pumain, R.; D'Arcangelo, G.; Tancredi, V. Network and

1778-1780.

pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro. Prog. Neurobiol. 2002, 68, 167-207.

[54]

Lampl, I.; Schwindt, P.; Crill, W. Reduction of Cortical Pyramidal Neuron Excitability by the Action of Phenytoin on Persistent Na+ Current. J. Pharmacol. Exp. Ther. 1998, 284, 228-237.

[31]

Dougalis, A.; Lees, G.; Ganellin, C. R. The sleep lipid oleamide

[55]

Spadoni, F.; Hainsworth, A. H.; Mercuri, N. B.; Caputi, L.;

[32]

may represent and endogenous anticonvulsant: an in vitro comparative study in the 4-aminopyridine rat brain slice model. Neuropharmacology 2003. Taylor, C. P. Na+ currents that fail to inactivate. Trends Neurosci.

[56]

Martella, G.; Lavaroni, F.; Bernardi, G.; Stefani, A. Lamotrigine derivatives and riluzole inhibit I NaP in cortical neurons. Neuroreport 2002, 13, 1167-1170. Kuo, C. C. A Common Anticonvulsant Binding Site for Phenytoin,

[33]

1993, 16, 455-460. Alzheimer, C.; Schwindt, P. C.; Crill, W. E. Modal gating of Na +

Carbamazepine, and Lamotrigine in Neuronal Na+ Channels. Mol. Pharmacol. 1998, 54, 712-721.

channels as a mechanism of peristant Na + current in pyramidal neurons from rat and cat sensorimotor coretx. J. Neurosci. 1993, 13, 660-673.

[57]

Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.; Swinyard, E. A. Anti-epileptic drug development. II. Anticonvulsant drug screening. Epilepsia 1978, 19, 404-428.

Voltage Gated Ion Channels: Targets for Anticonvulsant Drugs

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

29

[58]

Meldrum, B. Do preclinical seizure models preselect certain adverse effects of antiepileptic drugs. Epilepsy Res. 2002, 50, 33-

mechanism of action of PNU-151774E, a novel antiepileptic compound. J. Pharmacol. Exp. Ther. 1999, 288, 1151-1159.

533-535.

40.

[80]

Catterall, W. A. Structure and regulation of voltage-gated Ca 2+

[59]

McLean, M. J.; McDonald, R. L. Carbamazepine and 10,11-

comparison with carbamazepine. Epilepsia 2001, 42, 600-608.

190-199.

channels. Annu. Rev. Cell. Dev. Biol. 2000, 16, 521-555.

epoxycarbamazepine produce use and voltage dependent limitation

[81]

Ertel, E.; Campbell, K. P.; Harpold, M. M.; Hofmann, F.; Mori, Y.

of rapidly firing action potentials in mouse central neurons in cell culture. J. Pharmacol. Exp. Ther. 1986, 238, 727-738.

Nomenclature of voltage-gated calcium channels. Neuron 2000, 25,

[60]

Kuo, C. C.; Bean, B. P.; Chen, L. L.; Chen, R. C. Carbamzepine

[82]

Nowycky, M. C.; Fox, A. P.; Tsien, R. W. Three types of neuronal

calcium current reduction. Br. J. Pharmacol. 1990, 100, 800-806.

inhibition of neuronal Na + currents: quantitative distinction from phenytoin and possible clinical implications. Mol. Pharmacol. 1977, 51, 1077-1083.

[83]

calcium channel with different calcium agonist sensitivity. Nature 1985, 316, 440-443. Reuter, H. Calcium channel modulation by neurotransmitters,

[61]

Tecoma, E. S. Oxcarbazepine. Epilepsia 1999, 40, S37-S46.

enzymes and drugs. Nature 1983, 301, 569-574.

[62]

Bonifácio, M. J.; Sheridan, R. D.; Parada, A.; Cunha, R. A.;

[84]

Llinás, R. Yarom, Y. Electrophysiology of mammalian inferior

Patmore, L.; Soares-da-Silva, P. Interaction of the novel anticonvulsant, BIA 2-093, with voltage gated sodium channels:

[85]

olive neurones in vitro. Different types of voltage dependent ionic conductances. J. Physiol. 1981, 315, 569-584. McCleskey, E. W.; Fox, A. P.; Feldman, D. H.; Cruz, L. J.;

[63]

Leach, M. J.; Baxter, M. G.; Critchley, M. A. E. Neurochemical

Olivera, B. M.; Tsien, R. W.; Yoshikami, D. w -Conotoxin: direct

[64]

and behavioural aspects of Lamotrigine. Epilepsia 1991, 32, S4-S8. Leach, M. J.; Marden, C. M.; Miller, A. A. Pharmacological studies

and persistant blockade of specific calcium channels in nuerons but not muscle. Proc. Nat. Acad. Sci. U. S. A. 1987, 84, 4327-4331.

on Lamotrigine, a novel potential antiepileptic drug. II. Neurochemical studies on the mechanism of action. Epilepsia 1986, 27, 490-497.

[86]

Randall, A.; Tsien, R. W. Pharmacological dissection of multiple types of Ca 2+ channel currents in rat cerebellar granule neurons. J. Neurosci. 1995, 15, 2995-3012.

[65]

Miller, A. A.; Wheatly, P.; Sawyer, D. A.; Baxter, M. G.; Roth, B.

[87]

Jones, O. T. Ca2+ channels and epilepsy. Eur. J.Pharmacol. 2002,

Pharmacological studies on lamotrigine, a novel potential antiepiletic drug: I. Anticonvulsant profile in mice and rats.

[88]

447, 211-225. Chen, L.; El Husseini, A.; Tomita, S.; Bredt, D. S.; Nicoll, R. A.

[66]

Epilepsia 1986, 27, 483-489. Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.

Stargazin Differentially Controls the Trafficking of {alpha}- Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate and Kainate

Improved patch clamp techniques for high-resolution current recordings from cells and cell free memebrane patches. Pflug.

[89]

Receptors. Mol. Pharmacol. 2003, 64, 703-706. Coulter, D. A.; Huguenard, J. R.; Prince, D. A. Specific petit mal

[67]

Arch. 1981, 391, 85-100. Lees, G.; Leach, M. J. Studies on the mechanism of action of the

anticonvulsants reduce calcium currents in thalamic neurons. Neurosci. Lett. 1989, 98, 74-78.

novel anticonvulsant lamotrigine (Lamictal) using primary

[90]

Coulter, D. A.; Huguenard, J. R.; Prince, D. A. Differential effects

neurological cultures from rat cortex. Brain Research 1993, 612,

of petit mal anticonvulsants and convulsants on thalamic neurons:

[68]

Wang, C. M.; Lang, D. G.; Cooper, B. R. Lamotrigine effects on

S16.

[91]

Gomora, J. C.; Daud, A. N.; Weiergraber, M.; Perez-Reyes, E.

[69]

ion channels in neuronal cells. Epilepsia 1993, 34, 117-118. Ragsdale, D. S.; McPhee, J. C.; Scheuer, T.; Catterall, W. A.

Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol. Pharmacol. 2001, 60, 1121-1132.

Molecular determinants of state-dependent block of Na channels by

[92]

Gee, N. S.; Brown, J. P.; Dissanayake, V. U.; Offord, J.; Thurlow,

[70]

local anesthetics. Science 1994, 265, 1724-1728. Ragsdale, D. S.; McPhee, J. C.; Scheuer, T.; Catterall, W. A.

R.; Woodruff, G. N. The novel anticonvulsant drug, gabapentin (neurontin), binds to the alpha2delta subunit of a calcium channel.

Common molecular determinants of local anesthetic, antiarrhythmic and anticonvulsant block of voltage gated Na

[93]

J. Biol. Chem. 1996, 271, 768-776. Sutton, K. G.; Martin, D. J.; Pinnock, R. D.; Lee, K.; Scott, R. H.

[71]

channels. Proc. Nat. Acad. Sci. U. S. A. 1996, 93, 9270-9275. Shank, R. P.; Gardocki, J. F.; Vaught, J. L.; Davis, C. B.;

Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones. Br. J. Pharmacol. 2002,

Schupsky, J. J.; Raffa, R. B.; Dodgson, S. J.; Nortey, S. O.; Maryanoff, B. E. Topiramate: preclinical evaluation of a

[94]

135, 257-265. Stefani, A.; Spadoni, F.; Bernardi, G. Gabapentin inhibits calcium

[72]

structurally novel anticonvulsant. Epilepsia 1994, 35, 450-460. Gibbs, J. W. III; Sombati, S.; DeLorenzo, R. J.; Coulter, D. A.

currents in isolated rat brain neurons. Neuropharmacology 1998, 37, 83-91.

Cellular actions of topiramate: blockade of kainate-evoked inward

[95]

Fink, K.; Dooley, D. J.; Meder, W. P.; Suman-Chauhan, N.; Duffy,

currents in cultured hippocampal neurons. Epilepsia 2000, 41, S10-

S.; Clussman, H.; Göthert, M. Inhibition of neuronal Ca 2+ influx by gabapentin and pregabalin in the human neocortex.

[73]

Brown, S. D.; Wolf, H. H.; Swinyard, E. A.; Twyman, R. E.;

Neuropharmacology 2001, 42, 229-236.

White, H. S. The novel anticonvulsant topiramate enhances

[96]

Bayer, K.; Ahmadi, S.; Zeilhofer, H. U. Gabapentin may inhibit

[74]

GABA-mediated chloride flux. Epilepsia 1993, 34, 123-124. DeLorenzo, R. J.; Sombati, S.; Coulter, D. A. Effects of topiramate

synaptic transmission in the mouse spinal cord dorsal horn through a preferential block of P/Q-type Ca2+ channels.

on sustained repetitive firing and spontaneous recurrent seizure discharges in cultured hippocampal neurons. Epilepsia 2000, 41,

[97]

Neuropharmacology 2004, 46, 743-749. Zhang, X.; Velumian, A. A.; Jones, O. T.; Carlen, P. L. Modulation

S40-S44.

of high-voltage activated calcium channels in dentate granule cells

[75]

Zona, C.; Ciotti, M. T.; Avoli, M. Topiramate attenuates voltage-

by topiramate. Epilepsia 2000, 41, S52-S60.

gated sodium currents in rat cerebellar granule cells. Neurosci. Lett.

[98]

Stefani, A.; Spadoni, F.; Siniscalchi, A.; Benardi, G. Lamotrigine

[76]

1997, 231, 123-126. Taverna, S.; Sancini, G.; Mantegazza, M.; Franceschetti, S.;

inhibits Ca2+ currents in cortical neurons: functional implications. Eur. J. Pharmacol. 1996, 307, 113-116.

Avanzini, G. Inhibition of transient and persistent Na+ current

[99]

MacKinnon, R. Determination of the subunit stoichiometry of a

fractions by the new anticonvulsant topiramate. J. Pharmacol. Exp. Ther. 1999, 288, 960-968.

[100]

voltage-activated potassium channel. Nature 1991, 350, 232-235. Bezanilla, F. The voltage sensor in voltage-dependent ion channels.

[77]

Draguhn, A.; Jungclaus, M.; Sokolowa, S.; Heinemann, U.

Physiol. Rev. 2000, 80, 555-592.

Losigamone decreases spontaneous synaptic activity in cultured hippocampal neurons. Eur. J. Pharmacol. 1997, 325, 245-251.

[101]

Yellen, G. The voltage gated potassium channels and their relatives. Nature 2002, 419, 35-42.

[78]

Gebhardt, C.; Breustedt, J. M.; Noldner, M.; Chatterjee, S. S.;

[102]

MacKinnon, R. Potassium channels. FEBS Lett. 2003, 555, 62-65.

Heinemann, U. The antiepileptic drug losigamone decreases the

[103]

Zona, C.; Marchetti, C.; Margineau, D. G. The novel antiepileptic

[79]

persistent Na+ current in rat hippocampal neurons. Brain Res. 2001, 920, 27-31. Salvati, P.; Maj, R.; Caccia, C.; Cervini, M. A.; Fornaretto, M. G.;

drug candidate levetiracetam does not modify the biophysical porperties of the Na + channel in rat cortical neurons in culture. Society for neuroscience abstracts 1999, 25, 1868.

Lamberti, E.; Pevarello, P.; Skeen, G. A.; White, H. S.; Wolf, H.H.; Faravelli, L. Biochemical and electrophysiological studies on the

[104]

Zona, C.; Niespodziany, I.; Marchetti, C.; Klitgaard, H.; Bernardi, G.; Margineanu, D. G. Levetiracetam does not modulate neuronal

30

Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 1

Lees et al.

 

voltage-gated Na+and T-type Ca2+currents. Seizure 2001, 10, 279-

of KCNQ3 (c.925T-->C) in a Japanese family with benign familial

286.

neonatal convulsions. Ann. Neurol. 2000, 47, 822-826.

[105]

Madeja, M.; Georg Margineanu, D.; Gorji, A.; Siep, E.; Boerrigter,

685-690.

[121]

Vijai, J.; Kapoor, A.; Ravishankar, H. M.; Cherian, P. J.; Girija, A.

[106]

P.; Klitgaard, H.; Speckmann, E. J. Reduction of voltage-operated potassium currents by levetiracetam: a novel antiepileptic mechanism of action? Neuropharmacology 2003, 45, 661-671. Zona, C.; Tancredi, V.; Longone, P.; D'Arcangelo, G.; D'Antuono,

S.; Rajendran, B.; Rangan, G.; Jayalakshmi, S.; Mohandas, S.; Radhakrishnan et, a. Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population. Human Genetics 2003, 113, 461-463.

M.; Manfredi, M.; Avoli, M. Neocortical potassium currents are

[122]

Wallace, R. H.; Scheffer, I. E.; Barnett, S.; Richards, M.; Dibbens,

enhanced by the antiepileptic drug lamotrigine. Epilepsia 2002, 43,

L.; Desai, R. R.; Lerman-Sagie, T.; Lev, D.; Mazarib, A.; Brand, N.; Ben-Zeev, B.; Goikhman, I.; Singh, R.; Kremmidiotis, G.;

[107]

Zona, C.; Tancredi, V.; Palma, E.; Pirrone, G. C.; Avoli, M. Potassium currents in rat cortical neurons in culture are enhanced by the antiepileptic drug carbamazepine. Can. J. Physiol. Pharmacol. 1990, 68, 545-547.

Gardner, A.; Sutherland, G. R.; George, A. L. J.; Mulley, J. C.; Berkovic, S. F. Neuonal sodium-channel alpha1-subunit mutations in generalized epilepsy with febrile seizures plus. Am. J. Hum. Genet. 2001, 68, 859-865.

[108]

Brown, D. A.; Adams, P. R. Muscarinic supression of a novel

[123]

Spampanato, J.; Escayg, A.; Meisler, M. H.; Goldin, A. L.

[109]

voltage-sensitive K + current in a vertebrate neuron. Nature 1980, 283, 673-676. Halliwell, J. V.; Adams, P. R. Voltage clamp analysis of

Functional effects of two voltage gated sodium channel mutations that cause generalized epilepsy with febrile seizures plus type 2. J. Neurosci. 2001, 21, 7481-7490.

muscarinic excitation in hippocampal neurons. Brain Res. 1982,

[124]

Ito, M.; Nagafuji, H.; Okazawa, H.; Yamakawa, K.; Sugawara, T.;

[110]

250, 71-92. Wang, H. S.; Pan, Z.; Shi, W.; Brown, B. S.; Wymore, R. S.;

Mazaki-Miyazaki, E.; Hirose, S.; Fukama, G.; Mitsudome, A.; Wada, K.; Kaneko, S. Autosomal dominant epilepsy with febrile

Cohen, I. S.; Dixon, J. E.; McKinnon, D. KNCQ2 and KNCQ3 potassium channels subunits: molecular correlates of the M- channel. Science 1998, 282, 1890-1893.

[125]

seizures plus with missense mutations of the (Na + )-channel alpha 1 subunit gene, SCN1A. Epilepsy Res. 2002, 48, 15-23. Ceulemans, B. P.; Claes, L. R.; Lagae, L. G. Clinical correlations

[111] Robbins, J. KCNQ potassium channels: physiology,

of mutations in the SCN1A gene: from febrile seizures to severe

 

pathophysiology, and pharmacology. Pharmacol. Ther. 2001, 90,

myoclonic epilepsy in infancy. Pediatr. Neurol. 2004, 30, 236-243.

1-19.

[126]

Sugawara, T.; Tsurubuchi, Y.; Fujiwara, T.; Mazaki-Miyazaki, E.;

[112]

Charlier, C.; Singh, N. A.; Ryan, S. G.; Lewis, T. B.; Reus, B. E.; Leach, R. J.; Leppert, M. A pore mutation in an idiopathic epilepsy family. Nat. Genet. 1998, 18, 53-55.

mutation in neonatal human epilepsy. Science 1998, 279, 403-406.

Nagata, K.; Montal, M.; Inoue, T.; Yamakawa, K. Nav1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents. Epilepsy Res. 2003, 54, 201-207.

[113]

Biervert, C.; Schroeder, B. C.; Kubisch, C.; Berkovic, S. F.;

[127]

Sugawara, T.; Tsurubuchi, Y.; Agarwala, K. L.; Ito, M.; Fukuma,

[114]

Propping, P.; Jentsch, T. J.; Steinlein, O. K. A potassium channel

Schroeder, B. C.; Kubisch, C.; Stein, V.; Jensch, T. J. Moderate

G.; Mazaki-Miyazaki, E.; Nagafuji, H.; Noda, M.; Imoto, K.; Wada, K.; Mitsudome, A.; Kaneko, S.; Montal, M.; Nagata, K.; Hirose, S.; Yamakawa, K. A missense mutation of the Na+ channel

[115]

loss of function of cyclic-AMP-modulated KNCQ2/KNCQ3 K + channels causes epilepsy. Nature 1998, 396, 687-690. Rundfeldt, C. The new anticonvulsant retigabine (D-23129) acts as

alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction. Proc. Nat. Acad. Sci. U. S. A. 2001, 98, 6384-6389.

an opener of K+ channels in neuronal cells. Eur. J. Pharmacol. 1997, 336, 243-249. [116] Rundfeldt, C.; Netzer, R. The novel anticonvulsant retigabine activates M-currents in Chinese hamster ovary-cells tranfected with human KCNQ2/3 subunits. Neurosci. Lett. 2000, 282, 73-76.

[128]

Berkovic, S. F.; Heron, S. E.; Giordano, L.; Marini, C.; Guerrini, R.; Kaplan, R. E.; Gambardella, A.; Steinlein, O. K.; Grinton, B. E.; Dean, J.T.; Bordo, L. Hodgson, B.L.; Yamamoto, T.; Mulley, J.C.; Zara, F.; Scheffer, I.E. Benign familial neonatal-infantile seizures: characterization of a new sodium channelopathy. Ann.

[117]

Goldstein, S. A. N.; Bockenhauer, D.; O'Kelly, I.; Zilberberg, N. Potassium leak channels and the KNCK family of two-P-domain

[129]

Neurol. 2004, 55, 550-557. Wallace, R. H.; Scheffer, I. E.; Parasivam, G.; Barnett, S.; Wallace,

subunits. Nat. Rev. Neurosci. 2001, 2, 175-184.

G.

B.; Sutherland, G. R.; Berkovic, S. F.; Mulley, J. C. Generalized

[118]

Meadows, H. J.; Chapman, C. G.; Duckworth, D. M.; Kelsell, R.

epilepsy with febrile seizures plus: mutation of the sodium channel

E.; Murdock, P. R.; Nasir, S.; Rennie, G.; Randall, A. D. The

subunit SCN1B. Neurology 2002, 58, 1426-1429.

neuroprotective agent sipatrigine (BW619C89) potently inhibits the

[130]

Chioza, B.; Osei-Lah, A.; Nashef, L.; Suarez-Merino, B.; Wilkie,

864.

[119]

human tandem pore-domain K+ channels TREK-1 and TRAAK. Brain Res. 2001, 892, 94-101. Singh, N. A.; Westenskow, P.; Charlier, C.; Pappas, C.; Leslie, J.; Dillon, J.; Anderson, V. E.; Sanguinetti, M. C.; Leppert, M. F. KCNQ2 and KCNQ3 potassium channel genes in benign familial

H.; Sham, P.; Knight, J.; Asherson, P.; Makoff, A. J. Haplotype and linkage disequilibrium analysis to characterise a region in the calcium channel gene CACNA1A associated with idiopathic generalised epilepsy. Eur. J. Hum. Genet.: EJHG 2002, 10, 857-

neonatal convulssions: expansion of the functional mutation

[131]

Khosravani, H.; Altier, C.; Simms, B.; Hamming, K. S.; Snutch, T.

spectrum. Brain 2003, 126, 2726-2737.

P.;

Mezeyova, J.; McRory, J. E.; Zamponi, G. W. Gating effects of

[120]

Hirose, S.; Zenri, F.; Akiyoshi, H.; Fukama, G.; Iwata, H.; Inoue, T.; Yonetani, M.; Tsutsumi, M.; Muranaka, H.; Kurokawa, T.; Hanai, T.; Wada, K.; Kaneko, S.; Mitsudome, A. A novel mutation

mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J. Biol. Chem. 2004, 279, 9681-9684.