Voltage-Gated Sodium Channels in Neurological

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SCN8A. 12q13. Severe motor dysfunction. Cerebellar atrophy, ataxia, and mental ..... markedly improve the symptoms associated with this disease. [122,123].
CNS & Neurological Disorders - Drug Targets, 2008, 7, 000-000

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Voltage-Gated Sodium Channels in Neurological Disorders Mohamed Chahine*,1, Aurélien Chatelier1, Olga Babich2 and Johannes J. Krupp2 1

Le Centre de recherches Université Laval Robert-Griffard, and Department of Medicine, Laval University, Quebec City, Quebec, Canada 2

AstraZeneca R&D Södertälje, Molecular Pharmacology Department, Södertälje, Sweden Abstract: Voltage-gated sodium channels play an essential biophysical role on many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one -subunit and one or more -subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders.

Keywords: Neuronal excitability, action potential, splice variants, channelopathies, expression, toxins, local anesthetics, subunit specific blockers. INTRODUCTION The central nervous system (CNS) is the main component of the nervous system and is composed of the brain and the spinal cord. Information is transmitted along axons by action potentials (APs) caused by variations in membrane potentials. In most neurons the upstroke of such an AP is generated by a sodium conductance. It was Hodgkin and Huxley who not only recognized the importance of this sodium channel conductance for the upstroke of the neuronal AP, but also provided algorithms to describe the ionic basis of nerve excitability in the 1950’s, postulating distinct, independent molecular identities for sodium and potassium conductances [1,2]. This hypothesis was directly confirmed by Sakmann and Neher in the 1970’s when they recorded the first single channel elementary currents using the patchclamp technique [3,4]. The field of ion channel research took another giant step forward with the crystallization of several ion channels, pioneered by MacKinnon in the late 1990’s [5,6]. Today, the notion that the sodium channel conductance in neuronal cells is due to the opening and closing of individual molecular protein complexes, voltage-gated sodium channels (VGSC), is accepted within the scientific community. VGSC play a critical role in electrical signaling in the nervous system and are responsible for the initiation and propagation of APs. To fulfill such a function it is essential that VGSC enter first conducting and then non-conducting states rapidly (on a sub-ms to ms timescale) after an appropriate membrane depolarization. Recently, a number of mutations in VGSCs that often result in alterations of this rapid channel gating have been identified and linked to human diseases. Such channelopathies cause periodic paralysis, myotonia, long QT syndrome and other cardiac conductance *Address correspondence to this author at the AstraZeneca R&D Södertälje, Building 209, 15185 Södertälje, Sweden; Tel: +46 8 553 21686; Fax: +46 8 553 25440; E-mail: [email protected]

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disturbances, pain, and epilepsy. Considering these crucial physiological and pathological implications, it is not surprising that VGSC have been, and still are, key targets for drug discovery efforts by the pharmaceutical industry. This review focuses on this important class of ion channels, their involvement in neuronal disorders, and recent trends in the development of VGSC compounds for treatment of such disorders. STRUCTURE AND FUNCTION GATED SODIUM CHANNELS

OF

VOLTAGE-

VGSCs are composed of one -subunit, that forms the core of the channel and is responsible for voltage-dependent gating and ion permeation, and several auxiliary -subunits (Fig. 1) [7-9]. Whereas the -subunits are rather small proteins (22-36 kDa) that span the cell membrane only once, the -subunits are large proteins (~260 kDa) composed of four homologous domains (DI–DIV), with each domain containing six helical transmembrane-spanning segments (S1-S6). The S4 segments of each domain contain several polar amino acid residues that are part of the voltage sensor domain and are crucial for channel gating [10-12]. The short linkers connecting the S5 and S6 segments contain a short and nonhelical segment known as the P-segment. The four P-segments of an -subunit create the external mouth of the pore as well as the narrowest part of the ion pathway, the selectivity filter [1315]. The cytoplasmic loops connecting the four domains of VGSC have distinct functions. The large intracellular loop connecting homologous domains I and II possesses several shared protein kinase A (PKA), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK) phosphorylation sites and is therefore important in modulating sodium channels [16-19]. This region also interacts with A kinase anchoring protein 15 (AKAP15). AKAP15 enables rapid VGSC modulation by PKA [20]. Other proteins such as synaptotagmin can also interact with this loop [21]. The © 2008 Bentham Science Publishers Ltd.

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Fig. (1). Schematic representation of the topology of voltage-gated sodium channel subunits. Whereas the -subunit is a relatively small protein with only one transmembrane region, the pore-forming -subunit is large, containing four domains, each with six transmembrane regions. The fourth transmembrane region of each domain (S4, red) is highly positively charged and acts as the voltage-sensor. The channel pore is formed by the fifth and six transmembrane region and the connecting P-segment (S5,S6,P-segment, respectively; light blue) of each domain. Movement of the voltage-sensor is transmitted to the pore by the S4-S5-linker of each domain (blue). On the very right, a schematic view of the channel is shown, with color-coding as indicated.

cytoplasmic region between domains II and III can interact with ankyrin G, which plays a major role in inserting and localizing VGSC in the membrane [22-24]. The interaction occurs through a nine-amino-acid motif that is conserved in all VGSC ((V/A)P(I/L)AXXE(S/D)D) [23]. The interaction with ankyrin G also modulates the biophysical properties of VGSC [25]. The cytoplasmic linker between the domains III and IV is thought to act as a “hinged lid” that occludes the internal end of the permeation pathway during fast inactivation [10,26,27]. This loop also contains a PKC phosphorylation site that is involved in modulating VGSC activity [18,27]. The N and C terminals of VGSC also have sites that interact with cytoplasmic proteins such as annexin II [28] and calmodulin [29-31], respectively. In addition, the C terminal region takes part in VGSC fast inactivation [32,33]. THE -SUBUNIT To date, ten mammalian -subunit isoforms have been identified, nine of which are functional as VGSC (see Table 1). The tenth -subunit, called Nax or Nav2.1, is an atypical sodium channel as it lacks critical elements of VGSC functionality, like several positively charged amino acids needed for voltage-sensing. There is in fact no conclusive evidence that this -subunit is a functional ion channel. However, the protein appears to be able to sense the sodium concentration gradient across the membrane, and it has been proposed that Nax is critical for correct regulation of salt intake in animals [34,35]. Nax-KO animals indeed do have alterations in their

sodium homeostasis [35]. We will not further discuss Nax in the present paper. The remaining nine mammalian -subunits (Nav1.1 Nav1.9) are divided into two categories based on their sensitivity to the puffer fish toxin tetrodotoxin (TTX). Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, and Nav1.7 are sensitive to TTX, while Nav1.5, Nav1.8, and Nav1.9 are TTX-resistant. At least seven VGSC are mainly expressed in the nervous system. Nav1.1, Nav1.2, Nav1.3, and Nav1.6 are predominantly expressed in the CNS while Nav1.7, Nav1.8, and Nav1.9 are principally found in the peripheral nervous system (PNS) [36]. Small amounts of Nav1.7 and Nav1.9 transcripts have also been detected in the CNS [37-39]. Interestingly, there are species-differences in the expression of Nav1.7 in the CNS [40]. Nav1.4 and Nav1.5 are almost exclusively expressed in skeletal muscle and heart, respectively [41,42]. However, it recently has been reported that small amounts of Nav1.5 are also expressed in the CNS [43], and in skeletal muscle in animal models of critical illness myopathy [41]. Nav1.3 is widely distributed in embryonic neuronal tissue, but is strongly down-regulated postnatally [44]. However, Nav1.3 is up-regulated following nerve injury in adult rats [45-47]. THE -SUBUNIT Auxiliary -subunits are 22–36 kDa proteins with a single transmembrane segment, a long extracellular N-terminal with an immunoglobulin-like structure, and a short intracel-

Voltage-Gated Sodium Channels in Neurological Disorders

Table 1.

Protein Name

Nav1.1

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The -Subunits of the VGSC Superfamily Alternative/ Previous Protein Names

type I, Scn1a, rat I, HBSCI, GPBI

TTXSensitive

Yes

Predominant Location

CNS, PNS, DRG (strong)

Gene Symbol

SCN1A

Chromosomal Location (Human)

2q24

Phenotype of KO-Animal

Severe motor dysfunction and seizures

Global KO is lethal; Nav1.2

type II, rat II, HBSCII, HBA

Yes

CNS, DRG (weak)

SCN2A

2q23-24

SCN3A

2q24

SCN4A

17q23-25

Nav1.3

type III, rat III

Yes

CNS (embryonic), upregulated in DRG and spinal cord in some pain conditions

Nav1.4

SkM1, μ1

Yes

Skeletal muscle

Nav1.5

SkM2, H1, rH1

No

Heart muscle

SCN5A

3p21

Nav1.6

type IV, NaCh6, Na6, PN4, Scn8a, CerIII

Yes

CNS, PNS, glia, Nodes of Ranvier, DRG (strong)

SCN8A

12q13

Nav1.7

3

PN1, hNE, Nas

Yes

PNS, Schwann Cells, DRG (strong)

SCN9A

2q24

KO in brain causes perinatal death with severe hypoxia

Generalized epilepsy with febrile seizures (GEFS+); Severe myoclonic epilepsy in infancy (MEI); Familial hemiplegic migraine Benign familial neonatalinfantile seizures; Generalized epilepsy with febrile seizures (GEFS+)

Myotonia SCN5A–/– is lethal;

Congenital Long QT Syndrome type 3;

extensive myocardial fibrosis in aged SCN5A+/– KO

Progressive cardiac conduction defect (LenègreLev disease)

Severe motor dysfunction

Cerebellar atrophy, ataxia, and mental retardation

Global KO is lethal;

Familial Primary Erythromelalgia;

Inflammatory pain deficits in nociceptor-specific KO

Paroxysmal Extreme Pain Disorder;

Nav1.8

SNS, PN3, NaNG

No

PNS (sensory neurons), DRG (strong)

SCN10A

3p22-24

Deficits in pain perception

Nav1.9

SNS2, NaN, NaT, SCN12A

No

PNS, DRG (strong)

SCN11A

3p21-24

Peripheral imflammatory pain hypersensitivity

Nax

Nav2.1, Nav2.2, Nav2.3, SCL11, NaG

No

Heart, uterus, glia, PNS, smooth muscle, DRG (weak)

SCN6A (SCN7A)

2q21-23

Alterations in sodium homeostasis

lular C-terminal tail (Table 2). To date, four -subunits (1, 2, 3, and 4) have been identified (reviewed in [48,49]). The four subunits are divided into two groups. 1 and 3 associate non-covalently with the VGSC -subunit, whereas 2 and 4 bind covalently to the -subunit through a cysteine residue on the extracellular loop [50,51]. The association between one -subunit and one or more -subunits can modulate the channel gating and expression levels of VGSC [52-54]. Studies on the precise subunit composition of native VGSC thus have become a focus of recent efforts.

Known Human Channelopathies

Brugada Syndrom;

Congenital Indifference to Pain

INCREASED DIVERSITY THROUGH ALTERNATIVE SPLICING Alternative splicing is a mechanism for generating a versatile repertoire of different proteins from a common gene which occurs in numerous mammalian genes [55], including genes that encode mammalian VGSC. To date, four alternative splicing sites have been identified in mammalian VGSC -subunit genes [56-58], and alternative splicing happens also in at least one -subunit [59] (Fig. 2).

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Table 2.

Chahine et al.

The -Subunits of VGSC

Protein Name

Gene Symbol

Chromosomal Location (Human)

Phenotype of KO-Animal

Known Human Channelopathies

Growth retardation; 1

SCN1B

Spontaneous seizures.;

19q13.1

Altered sodium channel expression;

Generalized epilepsy with febrile seizures (GEFS+)

Altered nodal architecture; Prolonged QT and RR interval Defects in neuronal excitability; 2

SCN2B

11q23

3

SCN3B

11q23.3

4

SCN4B

11q23.3

Increased susceptibility to seizures; Arrythmia

The first site is located in the exon encoding part of segment S3 and all of S4 of domain I. The alternative splicing at this site appears to be developmentally regulated and occurs in exon 5 or 6, depending on the -subunit isoform. It generates two different transcripts containing either exon 5/6N (for neonatal) or 5/6A (for adult) that differ by one or two amino acids. This phenomenon was first described for the subunits of Nav1.2 [60,61] and Nav1.3 [61]. Transcripts containing exon 5N predominate in fetal and neonatal brain tissue, whereas exon 5N transcripts predominate in the adult brain. While similar transcripts have been identified in Nav1.6 [38,62] and Nav1.7 [38,63] exons, there is no evidence that the alternative splicing is developmentally regulated in these VGSC. Age-regulated spliced variants of exon 6 have also been observed for Nav1.5 [64,65]. However, no clear functional roles have been assigned to exons 5/6A and 5/6N. A recent study on Nav1.2 showed that the neonatal variant presents a hyperpolarized shift of inactivation accompanied by a faster time constant of fast inactivation and a slower recovery from fast inactivation [66]. These results

Congenital Long QT Syndrome type 3

may suggest that neonatal splice variants are less excitable than adult splice variants. However, in an as yet unpublished study we were not able to observe a significant difference in such parameters for the corresponding splice variants in Nav1.7 (Chatelier, Dahllund, Eriksson, Krupp and Chahine, unpublished). The second alternative splicing site is located in a region that encodes the cytoplasmic loop connecting transmembrane domains I and II. Depending on the -subunit, the alternative splicing leads to the insertion of amino acid sequences of varying length. The four splice variants in exon 12 of Nav1.3 (12v1, 12v2, 12v3, and 12v4) that have been characterized generate DI-II loops of different length [6769]. Interestingly, Nav1.1, Nav1.6, and Nav1.7 have two variants that correspond to the presence or absence of a relatively conserved 11-amino-acid sequence (V(I/K)IDK(A/P) (A/T)(T/S)DD(S/N); [38,67,70,71]. Very few studies have investigated the effects of alternative splicing events. While changes in intracellular loop length have been shown to in-

Fig. (2). Alternative splicing in voltage-gated sodium channel subunits. Exons with splicing events in the indicated subunits are indicated, as well as location of the splicing event in the full-length protein.

Voltage-Gated Sodium Channels in Neurological Disorders

fluence recovery from inactivation by Nav1.6 [71], changes in loop length have no marked effect on the biophysical properties of Nav1.3 [68]. It is interesting to note, however, that several phosphorylation sites are located in this loop [16-19]. Alternative splicing in this region could thus affect the modulation of VGSC via phosphorylation. The exon encoding the cytoplasmic region between domains II and III contains a third alternative 3’-terminal splicing site at a CAG/CAG motif that appears to be conserved in Nav1.5 and Nav1.8. These splice variants differ by the presence or absence of one glutamine residue [57,72]. While the functional role of this splicing event is unknown for Nav1.8, the presence of the glutamine decreases the peak current amplitude of Nav1.5 and shifts the steady-state inactivation by 7 mV toward a negative potential [72]. However, a common polymorphism of Nav1.5 (H558R) that reduces current density has no marked effect when the glutamine is absent [72]. Alternative splicing in this region can also result in the deletion of approximately 40 amino acids in Nav1.5 and Nav1.9, resulting in reduced current amplitude and altered channel gating for Nav1.5 [38,73]. Exons 18, which codes for the transmembrane segments S3 and S4 in domain III, is, like exon 5/6, a developmentally regulated alternative splicing site. Two alternatively spliced exons have been characterized for Nav1.6 (exon 18A and 18N). Exon 18N occurs in higher proportions in fetal brain tissue whereas exon 18A predominates later in life [70]. Exon 18N contains a stop codon that probably generates a truncated protein. Alternative splicing events in the exon coding for the 1 subunit generate two isoforms (1 and 1B). These variants differ in their C-terminal cytoplasmic domains but have similar tissue distributions [59]. While the functional impacts of these alternative splicing events on physiological functions are unknown, the fact that they occur in all VGSC indicates that they likely play an essential role in cell physiology. As such, a faulty regulation of the equilibrium between the proportions of splice variants may also result in pathological disorders. NEURONAL SUB-CELLULAR LOCALIZATION The localization of VGSC in neurons is a controlled phenomenon that leads to the production of specialized electrical domains like axon initial segments (AIS), axons, and dendrites. Since the -subunits have different biophysical properties, a particular sub-cellular localization may play a specific role in neuronal electrical activity such as AP initiation and propagation. In general, Nav1.1 and Nav1.3 have a somatodendritic localization (reviewed in [74]). It is therefore possible that they control the neuronal excitability threshold through postsynaptic potential integration. On the other hand, Nav1.2 and Nav1.6 seem to specialize in axonal conduction. Nav1.2 is present in unmyelinated axons, including the AIS and at immature nodes of Ranvier [75,76]. In mature nodes of Ranvier of myelinated axons, Nav1.2 is replaced by Nav1.6, which is also present in AIS and dendrites [76-78]. These localizations, together with their specific biophysical properties [79], indicate that Nav1.2 may be important for the conduction capabilities of unmyelinated axons, whereas Nav1.6 may be more adapted to saltatory conduction. It has been suggested that in the PNS Nav1.7 may localize to nerve

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endings [80]. This idea is supported by studies in differentiating PC12 cells where Nav1.7 localizes to axon growth cones [81]. Such a localization could help to explain the crucial role of this channel in nociceptive signalling [40,82,83]. BIOPHYSICAL PROPERTIES OF VOLTAGE-GATED SODIUM CHANNELS The gating of VGSC is regulated by the membrane potential and alterations of membrane potential can switch the channels between different main states: resting, activated, fast inactivated, and slow inactivated (Fig. 3). At membrane potentials more negative than –100 mV most VGSC are in a closed, resting state which could also be described as a not-activated and not-inactivated state. When the membrane potential reaches the activation threshold, it induces the transition from closed, resting to open, activated state. This activation state involves the outward movement of the positively charged transmembrane segments S4 with possible simultaneous rearrangements of the S2 and S3 segments, making the channel permeable to sodium ions [12,84,85]. The resulting rapid influx of sodium ions further depolarizes the cellular membrane. Within milliseconds after opening, VGSC enter a nonconducting inactivated state. Fast inactivation blocks sodium entry into the cell and so limits the membrane depolarization during an AP. The mechanism of this fast inactivation is associated with the occlusion of the intracellular mouth of the Na+ channel pore by the cytoplasmic loop linking domains III and IV of the  subunit [27,86]. The carboxy terminal domain of the channel also takes part in the inactivation process by stabilizing the inactivation gate and minimizing channel reopening [32,33]. Inactivation of sodium channels facilitates repolarization of the neuronal cell membrane to more hyperpolarized potentials, with a subsequent return of the VGSC to the closed, resting state. This return to an activable state is necessary for the generation of the next AP. The refractory period of AP generation is determined by the speed of recovery from fast inactivation, which is modulated by the membrane voltage and can occur on a timescale between milliseconds and seconds. In contrast to slow inactivation (see below), fast inactivation is rather insensitive to changes in extracellular sodium concentration. However, multivalent transitional metal ions binding at the selectivity-related region of the outer pore profoundly slows fast inactivation [87]. Considering that the fast inactivation gate is located at the intracellular part of the pore this gating modulation suggests allosteric coupling of “pore events” with subtle conformational changes that alter the fast inactivation process. Thus, sodium channel inactivation and ion permeation are complex but not necessarily separate processes. Slow inactivation is kinetically (time scale of seconds rather than milliseconds) and structurally distinct from fast inactivation. The slow inactivated state occurs in neurons after high frequency firing or during prolonged membrane depolarization and can therefore decrease VGSC availability leading to lower membrane excitability. Block of the fast inactivation gate by specific antibodies [88] or mutations that eliminate fast inactivation do not affect slow inactivation

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Fig. (3). Schematic representation of the main gating states of voltage-gated sodium channels. From a closed, resting state, channels open through outward movement of the voltage-sensor, which is through the S4-S5 linker is transmitted to the channel pore. Fast inactivation occurs in large parts through occlusion of the intracellular mouth of the channel by the DIII-DIV cytoplasmic linker. The molecular movements that cause slow inactivation are less well understood, as indicated by the questionmark. Slow inactivation can develop from the closed state, as outlined in the text.

[89]. Numerous studies link the pore-lining segments of Na+ channels as the site of a conformational change during slow inactivation rather than the cytoplasmic region of the channel. For example, mutations in the selectivity filter as well as in the outer-carboxylates ring affect slow inactivation suggesting a close relationship between the permeation pathway and the slow inactivation gate [90-93]. In contrast to the Ctype inactivation of potassium channels, in which a collapse of the outer pore-forming segments occludes the aqueous permeation pathway [94], the external permeation pathway to the selectivity filter in Na+ channel appears to remain open during slow inactivation [95]. The kinetics and steady-state levels of slow inactivation are influenced by the concentration of extracellular alkali metal cations [96]. Raising sodium concentration from 10 to 150 mM increases the rate of recovery from slow inactivation at hyperpolarized voltages, decreases the rate of slow inactivation at depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. This data correlates well with a Na+-saturated model [97] that predicts a stabilizing effect of the conducting pore conformation by Na+ ions binding between neighboring domains in the outercarboxylates ring of the pore. The slow inactivation process however is not limited to the pore and involves many other regions of the channel [98,99]. Slow inactivation can also develop from the closed state before the channel reaches the conducting open state [100102]. Closed-state inactivation is more likely to occur during slow, small depolarizations prior to channel opening, during which occupancy of the partially activated closed states is prolonged. In a revised Na channel inactivation model Armstrong suggested that closed-state inactivation requires only S4 activation of domain III and IV, which does not open the activation gate [102]. Differences in the development of closed-state inactivation between different -subunits may have important physiological implications. For example, closed-state inactivation of Nav1.7 is slow compared to the Nav1.6 subunit [103], such that even after a long sub-

threshold depolarization a significant number of Nav1.7 channels are able to transition into the open, activated state. Together with a presumed localization of Nav1.7 in peripheral nerve endings (see above) this feature make this channel a likely candidate for the threshold channel that converts a passive generator potential in the periphery into an actively propagated AP. VOLTAGE-GATED SODIUM CHANNELS IN NEUROLOGICAL DISORDERS Considering the essential role of VGSC in the generation of the upstroke of the neuronal AP, it is not surprising that their dysfunction can cause severe neurological disorders. Indeed, many clinically used psychopharmaca, like carbamazepine, phenytoin, or lamotrigine, owe their positive effects, at least partially, to their inhibition of VGSC. Many other classical and novel antiepileptic drugs also share the same mechanism of action [104,105]. Moreover, due to their finely tuned biophysical properties, even minor alterations in the protein structure of VGSC can have dramatic consequences, as best illustrated by the dysfunctions caused by inherited mutations in VGSC. Such channelopathies can cause several epileptic phenotypes like generalized epilepsy with febrile seizures or benign familial neonatal infantile seizures; several inherited pain disorders, familial hemiplegic migraine, as well as other psychiatric disorders. The following paragraph will discuss aspects of these channelopathies. EPILEPSY Epilepsy is one of the most prevalent neurological disorders in the world today. It consists of a group of brain diseases with a wide range of clinical features and causes. However, all share seizure as a common behavioral phenotype. Many hypotheses have been proposed to explain the development of seizures. In recent years, genetic discoveries have revealed the central role of ion channels in the pathophysiology of idio-

Voltage-Gated Sodium Channels in Neurological Disorders

pathic epilepsies. Monogenically inherited epileptic syndromes are associated with mutations in genes that code for subunits of voltage-gated and ligand-gated ion channels. In the case of voltage-gated ion channels, Na, K, and Cl channel mutations have been associated with forms of generalized epilepsy and infantile seizure syndromes. In fact, febrile seizures affect 3% of children under six years of age. Generalized epilepsy with febrile seizures plus (GEFS+) is a term used to describe patients who have seizures with fever that persist beyond six years of age. The first locus correlated with GEFS+ was identified on chromosome 19q13.1 of an Australian family [106]. This chromosome also harbors the SCN1B gene, which encodes the 1-subunit of the Na channel and plays an important regulatory role. While SCN1B is also expressed in heart and skeletal muscle tissue, these GEFS+ patients do not suffer from periodic paralysis, myotonia, or arrhythmia. Further studies will be required to elucidate this phenomenon. A C (cytosine) to G (guanine) mutation at position 387 on SCN1B substitutes a highly conserved cysteine residue with a tryptophan (C121W) [106]. The cysteine is thought to be involved in forming a disulfide bridge and thus to stabilize the immunoglobulin-like structure of the extracellular region of the 1-subunit. Functional expression experiments in Xenopus laevis oocytes showed that fast inactivation is disrupted in the presence of this particular mutation, which is consistent with a loss-of-function mutation. While it may seem paradoxical that a loss-of-function of the 1-subunit gives rise to a gain-of-function of the channel, it is the gain-of-function of the channel that causes the disease. Interestingly, it was found that VGSC expressing the mutated 1-subunit are less sensitive to the anticonvulsant phenytoin [107]. A five-amino-acid deletion (I70-E74del) on the same extracellular Ig domain of the 1 subunit has also been identified in a patient with a GEFS+ clinical phenotype [108]. The biophysical characterization of the deletion mutant in Xenopus laevis oocytes showed that it causes slowly inactivating Na currents, which is consistent with a sodium channel gain-of-function. Over 150 mutations have been identified in patients with epilepsy. Many are nonsense and exhibit the haploinsufficiency for SCN1A. The majority of GEFS+-causing mutations in the SCN1A gene are gain-of-function mutations, that induce a persistent inward sodium current or shift inactivation towards more positive potentials. However, there are also reports in the literature that some mutations result in loss-of-function.

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(BFNIS). The L1563V splice variant causes a gain-offunction in neonates but has no effect in adult splice variant [66]. This may explain why patients with the L1563V mutation have more seizures in infancy and points to another role for splice variants in the pathology of epilepsy. Severe myoclonic epilepsy in infancy (SMEI) has been mapped to SCN1A. Certain mutations in this gene abolish channel function, which is consistent with the notion that loss-of-function and haploinsufficiency for SCN1A cause SMEI. A recently developed animal model should help identify other factors that play a role in SMEI [110,111]. PAIN DISORDERS As outlined above, several VGSC are almost exclusively expressed in nociceptive pathways of the PNS, making them likely to have important physiological functions in the perception of pain. The TTX-resistant VGSC Nav1.8 and Nav1.9 are two cases in point. There is extensive evidence for involvement of both of these channels in different pain conditions. For example, the intrathecal administration of antisense oligonucleotide to Nav1.8 has been shown to be analgesic in animal models for neuropathic pain [112]. Nav1.8 is also likely to be involved in chronic inflammatory pain [113]. Although Nav1.8 KO mice were previously shown to have rather minor deficits in pain perception [114], a recent elegant study on the Nav1.8 KO mice has pointed to an important role of this channel in cold induced pain [115]. The TTX-sensitive VGSC Nav1.3 is highly expressed in embryonic brain and sensory neurons, but is down-regulated in most adult neuronal tissue [44]. However, expression of Nav1.3 is up-regulated in adult DRGs following axotomy or chronic constriction injury (CCI) of the sciatic nerve in adult rats [116]. Because Nav1.3 channels are rapidly repriming this up-regulation has been suggested to underlie the TTXsensitive ectopic discharges associated with axonal damage [117]. Another TTX-sensitive VGSC, Nav1.7, has recently been shown to be central in several pain conditions. As already mentioned, this channel exhibits a slow repriming and slow closed-state inactivation. Together with the presumed localization in peripheral nerve endings, this makes Nav1.7 uniquely suited to amplify small excitatory inputs generated by sensory ion channels in the periphery into actively propagated APs, i.e. act as the threshold channel.

SCN1A is not the only gene linked to GEFS+. A SCN2A mutation (R187W) affecting the S2-S3 cytoplasmic linker, which is highly conserved, has been identified in a Japanese patient with a GEFS+ clinical phenotype [109]. The R187W mutation slows the kinetics of inactivation and shifts the steady-state inactivation toward more negative potentials without affecting conductance. These biophysical dysfunctions could underlie the neuronal hyperexcitability that triggers seizures.

Indeed, in the last four years numerous studies have linked mutations in this channel to several inherited human pain disorders (Fig. 4). Primary erythromelalgia (also called erythermalgia) was the first inherited human disease to be linked to mutations in Nav1.7 [118,119]. This dominantlyinherited chronic disease is characterized by edema, redness, warmth, and bilateral pain, predominantly in the extremities. The identified mutations lead to hyperactivity of Nav1.7, resulting in repetitive firing in the peripheral nerves, likely to cause pain even in the absence of external painful stimuli [120,121]. VGSC blockers (lidocaine and mexiletine) can markedly improve the symptoms associated with this disease [122,123].

A number of mutations of the SCN2A gene (R223Q, V891I, L1003I, R1319Q, L1330F, and L1563V) have been reported to cause benign familial neonatal-infantile seizures

Another rare inherited disease, paroxysmal extreme pain disorder, has also recently been linked to mutations in Nav1.7 [124,125]. Burning rectal, ocular, and submandibular pain

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Fig. (4). Position of mutations in Nav1.7 that lead to congenital clinical phenotypes in a topology model (left) and a schematic protein view (right). As indicated in the schematic view on the right, mutations leading to erythromelalgia (white circles) cluster primarily in the S4S5 linker of DI and DII, as well as the channel pore and the voltage sensor, in agreement with primary effects on activation kinetics with such mutations. In contrast, mutations that lead to paroxysmal extreme pain disorder (yellow circles), cluster in the S4-S5 linker of DIII and DIV, and the DIII-DIV cytoplasmic loop, causing impaired inactivation. Mutations leading to congenital indifference to pain are either homozygous stop codon mutations (pink flashes) or compound heterozygous mutations (pink stars). All of these mutations are likely to cause non-functional Nav1.7 proteins.

and flushing are typically associated with this disease. The identified mutations all cause hyperactivity of the channel through impaired inactivation of Nav1.7. Even further evidence for an important role of the Nav1.7 sodium channel in nociception stems from three recently published studies on the very rare phenotype of congenital inability to sense pain [40,82,83]. Individuals with this disease are not able to perceive any form of pain, although all other sensory modalities appear normal. Although the three studies describe different families with this disease, they all link the phenotype to distinct nonsense or stop-codon mutations in SCN9A, the gene encoding Nav1.7. As expected, the mutations were shown to lead to non-functional Nav1.7 proteins when expressed in HEK293 cells [40,82]. For one of the cases it was also shown that the truncated protein does not affect endogenous voltage-gated sodium currents in a neuroblastoma cell line [40] (Fig. 5). Interestingly, the findings in humans are in some contrast to data from a global Nav1.7-null mutant in mice, where it causes a lethal phenotype shortly after birth [80]. Profound deficits in pain behavior can however be observed in mice when Nav1.7 is knocked down in nociceptive neurons only [80]. These differences in the lethality of Nav1.7 deficiency between humans and rodents may be due to differences in the central expression of Nav1.7 [40].

FAMILIAL HEMIPLEGIC MIGRAINE While familial hemiplegic migraine (FHM) is usually associated with mutations in the Cav1.2 calcium channel, a rare type of FHM has been linked to a Q1489K mutation in the SCN1A gene, which encodes the -subunit of Nav1.1 [126]. This gene, has also been associated with epilepsy (see above). The mutation occurs in a region that is important for Na channel inactivation and causes a much faster recovery from inactivation, a shift of steady-state activation, a normal steady-state inactivation, and also affects slow activation. More importantly, the authors also reported that there is a 3% increase in the persistent sodium current. OTHER PSYCHIATRIC DISORDERS The rapid pace of discoveries suggests that Na channel mutations are significant factors in the etiology of neurological diseases and may contribute to psychiatric disorders as well [127]. Given the essential role of Na channels in the generation of APs and neuronal firing, it is legitimate to speculate that mutations in VGSC may be responsible for more complex psychiatric disorders. In fact, it has recently been shown that splice variants in or adjacent to SCN8A, which encodes the Nav1.6 isoform expressed in the CNS and the PNS, are involved in cerebellar atrophy, ataxia, and mental retardation [128]. One mutation produces a stop codon, that truncates parts of domain IV and the C-terminal, which

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Fig. (5). Functional effects of a stop-codon mutation (Y328X) leading to congenital indifference to pain. The mutation does not produce functional protein, nor is there an apparent effect on endogenous voltage-gated sodium channels in a neuroblastoma cell line (figure reproduced with permission from [40]).

are essential for sodium channel function. It has been suggested that haploinsufficiency and reduced levels of Nav1.6 in heterozygous individuals may reduce neuronal excitability and cause altered firing patterns. PHARMACOLOGY OF VOLTAGE-GATED SODIUM CHANNELS Considering the important function of VGSC in AP generation and propagation it is not surprising that numerous plant and animal species produce biological toxins for predatory or protective purposes that modify the properties of

VGSCs. Likewise, numerous early pharmacological agents that were cornerstones in the development of the pharmaceutical industry have been shown to affect VGSCs, mostly in a subunit-unspecific manner. And, as outlined above, the last decade or so has seen ever increasing evidence in the literature for important contributions of different VGSC isoforms to pathological situations. This, together with the recent development of new screening technology, has rekindled a strong interest within the pharmaceutical industry for small molecules targeting VGSCs. The present focus within the industry is on subunit-specific compounds and/or com-

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Table 3.

Chahine et al.

Receptor Sites of Neurotoxins on VGSC

Receptor Site

Toxins

Predominant Effect on VGSC

Predominat Sites for Interaction

Sodium current block

IS2-6, IIS2-6, IIIS2-6, IVS2-6

Persistent channel activiation

IS6, IVS6

Slowing of inactivation

IS5-S6, IVS3-S4, IVS5-S6

Shift of activation to more negative potentials

IIS1-S2, IIS3-S4

Shift of activiation to more negative potentials

IS6, IVS5

Delay or inhibition of inactivation

IVS3-S4

Sodium current block

IS6, IIIS6, IVS6

Tetrodotoxin 1

Saxitoxin μ-conotoxins Veratridine Grayanotoxin

2

Aconitin Batrachotoxin

3 4

 -scorpion toxin Sea anemone toxins  and -scorpion toxins Brevetoxins

5

Ciguatoxins

6

-conotoxins Local anaesthetics

LA-site

Antiarrythmics Antiepileptics

pounds that interact with VGSCs with defined mechanismbased activity. Specifically, compounds that have increased apparent affinity for the slow inactivated state of VGSC are thought to be useful for therapeutic purposes. BIOLOGICAL TOXINS The effects and binding properties of biological toxins that interact with VGSCs have been studied extensively. Based on such studies several toxin binding sites can be distinguished (Table 3). The puffer fish toxin TTX and the paralytic shellfish poison saxitoxin (STX) 1 interact with high affinity (Kd of 1-10 nM) with the outer vestibule of the channel, thereby blocking ion flux through VGSC. Numerous residues just above the selectivity filter are thought to interact with both toxins, with a single residue determining TTX-sensitivity of the different VGSC -subunits [129]. Several polypeptides produced by snails of the Conus family also block VGSCs, and a subset of them (the μconotoxins) do so by acting at the same site as TTX and STX. However, because conotoxins are considerably larger than TTX or STX, additional sites on the channel determine binding affinity. Interestingly, several recent reports indicate some subunit selectivity among the different conotoxins [130,131]. Scorpion toxins also modulate VGSC functionality. These toxins interact with VGSC predominantly at the voltage-sensing regions, with -scorpion toxins slowing inactivation and -scorpion toxins affecting activation. Like for the conotoxins, several scorpion toxins show considerable 1

Note that both toxins are accumulated to deadly concentrations by the named host species, but are not actually produced by these species. TTX is produced by bacteria and accumulated by the puffer fish predominantly in their ovaries and liver. Likewise, STX is produced by marine plankton Dinoflagellata species, but accumulated by plankton-feeding species like mollusks, arthropods, echinoderms, and some fish.

subunit selectivity [132,133]. The site of action for scorpion toxins is also the site of action for sea anemone toxins (ATX) and some funnel-web spider toxins. Some species of South American arrow poison frogs secret batrachotoxin (BTX), a toxin that persistently activates VGSC. The site of interaction appears to span several domains of the channel, including some regions that form part of the channel pore. Veratridine also interacts with this site and also persistently activates the channel. It has been suggested that both toxins actually bind within the channel lumen close to the site for local anesthetics [134]. Indeed, veratridine has been shown to activate a rudimentary channel consisting of only the pore-forming regions of a VGSC [135]. The flower heads of certain Chrysanthemum species produce pyrethrins, which serve as blueprints for the synthesis of related pyrethroids. These compounds prolong the sodium current in excitable membranes, making them powerful neurotoxins. Because the compounds show considerable selectivity between mammalian and invertebrate VGSC, they are commercially highly interesting as insecticides [136]. Several marine dinoflagellate species produce different lipid-soluble polyethers toxins, like brevetoxins and CTX, that affect activation and inactivation of VGSCs. The site of interaction is somewhat less defined, but during binding the toxin backbone is in the vicinity of the S5-S6 extracellular loop of domain IV [137]. The above list of natural toxins affecting VGSC is by no means complete, but is barely a brief glimpse of the enormous complexity of this extensive and exciting field. For example, recently four tarantula toxins have been described that show subunit-selective modulation of VGSC [138]. We refer the interested reader to more extensive reviews on natural toxins that affect VGSC functionality [139-141].

Voltage-Gated Sodium Channels in Neurological Disorders

SMALL MOLECULE BLOCKERS: LEGACY COMPOUNDS VGSC blockers were developed and used for therapeutic purposes long before the molecular targets were identified or cloned, or before a mechanistic understanding of the molecular action of such compounds was developed. One of the earliest compounds used for therapeutic purposes, that was later shown to block VGSC, is cocaine. Cocaine, an aminoester, was the first local anesthetic drug useful in clinical surgery. It was also soon realized that the anesthetic properties of cocaine were preserved in chemically similar structures that had less undesirable side effects. This quickly led to the development of an entire class of related compounds comprising other aminoesters like benzocaine and procaine, as well as aminoamides, like bupivacaine and lidocaine (Fig. 6). Most of these drugs were/are applied topically or intrathecally, thereby minimizing adverse side effects. However, some of these compounds have also been used in the clinic with systemic application. For example, intravenous lidocaine has shown efficacy in numerous clinical trials on neuropathic pain conditions [142,143]. In neuropathic pain models in the rat a peripheral site of action for lidocaine has been proposed [144]. The concept that such a peripheral site of action might also contribute to the efficacy in human clinical trials is supported by the fact that lidocaine patches are not only used for anesthetic, but also analgesic purposes [123].

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curs. Accumulation occurs because the inter-stimulus interval is too short to allow complete unbinding and exit of the blocker from the channel. This means that there is a negative correlation between length of inter-stimulus interval and degree of accumulation of drug-bound, blocked channels. Thus, assuming that all other parameters like holding and test potentials, drug concentration, etc. are equal, the percentage of channels blocked will directly depend on the frequency of stimulation. Although this phenomenon is often referred to as ‘use-dependent block’ the term ‘frequencydependent block’ thus appears a much better term. For further discussion on the mechanism of interaction of LA with VGSC we refer the reader to [151,152].

The mechanism by which local anesthetics (LA) like bupivacaine, lidocaine, tetracaine, or etidocaine interact with VGSCs has been the subject of a plethora of scientific studies. It was Catterall’s lab that firmly established that the interaction site (LA-site) is located in the channel lumen between selectivity filter and intracellular channel gate. Using the approach of alanine-scanning mutagenesis they showed that most of the interactions of LA with VGSC occur with residues in the S6 regions of domains III and IV [145-148]. The interaction of LA with VGSC are complex : in the resting state VGSC have low affinity for LA, but upon channel activation a considerable increase in apparent affinity for these compounds occurs. In theory this difference could arise as a change in the conformation of the binding site during channel gating, i.e. a state-dependent change in affinity, or as a change in accessibility of the binding site upon gating, i.e. a state-dependent change in access to the binding site. Indeed, two hypotheses have been proposed to explain the complex interaction of local anesthetics (LA) with VGSC: (1) the modulated-receptor hypothesis [149], proposing that LA bind to open and inactivated channels with higher affinities than to the resting/closed channels, and (2) the guarded receptor hypothesis [150], proposing that LA are bound with a constant affinity but that the state-dependent access results in the apparent differences in binding kinetics. Both models explain very well some of the experimental results. As often in science, the truth may lie somewhere in between, and it is not unreasonable to think that the shift in the apparent affinity of LA during channel gating is due to a combination of both hypotheses. A further complication in the interpretation of experiments with LA on VGSC is the fact that accumulation of drug-bound, blocked channels with repeated stimulation oc-

Fig. (6). Chemical structure of some legacy compounds active on voltage-gated sodium channels.

Besides the "caine" class of analgesic compounds, certain clinically used anticonvulsants, antidepressants, and antiarrythmics have inhibitory activity on VGSCs, that at least partially underlies their clinical efficacy. Among such compounds are phenytoin [108], carbamazepine [153], fluphenazine [154], and amitriptyline [144].

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SMALL MOLECULE BLOCKERS: RECENT TRENDS In the last decade the field of VGSC has seen a renaissance within the pharmaceutical industry, primarily triggered by the advent of new screening technologies. These efforts yield an ever growing number of new compounds that are being reported within the scientific literature. Among them a few compounds should be highlighted as important milestones in a development that almost certainly has not yet seen its full peak.

Chahine et al.

cellular and subcellular expression is highly controlled, and often determined by the subunit combination, or the specific splice variant expressed. Due to their essential physiological role in the initiation and propagation of the AP, a variety of natural toxins interfere with VGSC functionality. Furthermore, numerous neurological pathologies are caused by dysfunction of VGSCs. Most dramatically this is evident in the neurological diseases caused by channelopathies of VGSC.

Co-102862 (also V-102862) is the most interesting compound in a group of compounds described by researchers from Purdue that have potent anticonvulsant activity in rat seizure models [155,156] (Fig. 7). This compound has a much higher state-dependence than known for previous sodium channel blockers [157], resulting in a dissociation constant of 600 nM for the inactivated state of the rat Nav1.2 channel, as compared to a dissociation constant of >15 μM for the resting state. In the presence of the compound the steady-state inactivation curve is thus shifted in the hyperpolarizing direction, which reduces the fraction of channels available for activation at physiologically relevant membrane voltages. Although Co-102862 is not a subunit-specific sodium channel inhibitor, it appears that this compound as well as a further improved follow-up compound, PPPA [158], have been advanced into human clinical trials [159], although no information on the outcome of these studies is available. Another interesting compound that recently was described is the Merck compound CDA-54 [160]. This peripherally restricted compound is a potent, state-dependent blocker of VGSCs. The compound interacts with both Nav1.7 and Nav1.8 with high affinity in the inactivated state, but since the compound has similarly high activity at Nav1.2 and Nav1.5, it is not a subunit-selective sodium channel blocker. However, there appears to be a significant selectivity over voltage-gated calcium channels [161]. Due to its strong state-dependency, CDA-54 is efficious in preclinical animal models of neuropathic pain with no effect on acute nociception or motor coordination at effective doses. Likewise, no effects on various cardiac parameters were seen in anesthetized dogs at CDA-54 plasma concentrations up to 6.7 μM [161]. Recently, researchers from Merck have described another group of compounds acting as non-selective VGSC inhibitors, as exemplified by BNZA in Fig. 7 [162]. Recently, researchers from Abbott and Icagen have described the subtype-selective Nav1.8 compound A-803467 [163]. This small molecule appears to be the first truly isoform selective voltage-gated sodium channel blocker. The compound has been described to inhibit the inactivated Nav1.8 channel half-maximally at a single-digit nanomolar concentration, while super-micromolar concentrations of A803467 are required to inhibit other voltage-gated sodium channel isoforms [163]. The compound was efficacious in the Chung and CCI models of neuropathic pain, with improved safety margins, as compared to other sodium channel blockers, in the locomotor and rotarod tests. CONCLUSION VGSC are beautifully complex creatures that exhibit intricate biophysical characteristics. On a molecular level their

Fig. (7). Chemical structure of some recently described blockers of voltage-gated sodium channels.

Due to their critical role in neuronal and cellular excitability VGSC have been the focus of intensive research efforts for decades. They also are at the center of considerable drug discovery efforts. However, despite all these efforts there are still considerably large gaps in our understanding of VGSC. The precise nature of the molecular gating movements in VGSC is still enigmatic. When will we see the first crystal structure of a VGSC? What is the subunit composition of native VGSC in different tissues? And how exactly do some widely used pharmaceutical compounds interact with VGSC? Clearly: much remains to be learned. REFERENCES [1] [2] [3]

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Received: February 11, 2008

Accepted: February 21, 2008

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