[William Catterall (centre) is Professor and Chair of Pharmacology, John Oakley (right) is Acting Assistant Professor of Neurology, and Franck Kalume (left) is Acting Instructor of Pharmacology at the University of Washington School of Medicine in Seattle. Together with colleagues, they have produced and characterized a mouse model of severe myoclonic epilepsy of infancy (SMEI), which exhibits all of the characteristics of the human disease. Their work reveals that mutations of NaV1.1 channels in this disease cause selective loss of sodium current and electrical excitability of GABAergic inhibitory neurons, which is likely to be responsibility for both epilepsy and co-morbidities in SMEI.]
Voltage-gated sodium channels initiate action potentials in brain neurons, and sodium channel blockers are used in therapy of epilepsy. Mutations in sodium channels are responsible for genetic epilepsy syndromes with a wide range of severity, and the NaV1.1 channel encoded by the SCN1A gene is the most frequent target of mutations. Complete loss-of-function mutations in NaV1.1 cause severe myoclonic epilepsy of infancy (SMEI or Dravet's Syndrome), which includes severe, intractable epilepsy and comorbidities of ataxia and cognitive impairment. Mice with loss-of-function mutations in NaV1.1 channels have severely impaired sodium currents and action potential firing in hippocampal GABAergic inhibitory neurons without detectable effect on the excitatory pyramidal neurons, which would cause hyperexcitability and contribute to seizures in SMEI. Similarly, the sodium currents and action potential firing are also impaired in the GABAergic Purkinje neurons of the cerebellum, which is likely to contribute to ataxia. The imbalance between excitatory and inhibitory transmission in these mice can be partially corrected by compensatory loss-of-function mutations of NaV1.6 channels, and thermally induced seizures in these mice can be prevented by drug combinations that enhance GABAergic neurotransmission. Generalized epilepsy with febrile seizures plus (GEFS+) is caused by missense mutations in NaV1.1 channels, which have variable biophysical effects on sodium channels expressed in non-neuronal cells, but may primarily cause loss of function when expressed in mice. Familial febrile seizures is caused by mild loss-of-function mutations in NaV1.1 channels; mutations in these channels are implicated in febrile seizures associated with vaccination; and impaired alternative splicing of the mRNA encoding these channels may also predispose some children to febrile seizures. We propose a unified loss-of-function hypothesis for the spectrum of epilepsy syndromes caused by genetic changes in NaV1.1 channels, in which mild impairment predisposes to febrile seizures, intermediate impairment leads to GEFS+ epilepsy, and severe or complete loss of function leads to the intractable seizures and comorbidities of SMEI.
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The epilepsies are a heterogeneous group of conditions characterized by recurrent seizures. While many pathophysiological changes contribute to seizure susceptibility, recent work suggests that genetic factors are important. Polygenic inheritance patterns have been associated with febrile seizures and may be important in determining susceptibility to acquired epilepsy following brain injury. Monogenic inheritance patterns are seen in a number of epilepsies associated with mutations in ligand- or voltage-gated ion channels. The gene most frequently associated with epilepsy is SCN1A, which codes for the α subunit of the Nav1.1 sodium channel.
Voltage-gated sodium channels
Voltage-gated Na+ channels in the brain are complexes of a 260 kDa α subunit in association with auxiliary β subunits (β1–β4) of 33–36 kDa (Catterall, 2000). The α subunit contains the voltage sensors and the ion-conducting pore in four internally repeated domains (I–IV), which each consists of six α-helical transmembrane segments (S1–S6) and a pore loop connecting S5 and S6 (Catterall, 2000). The β subunits modify the kinetics and voltage dependence of gating and serve as cell adhesion molecules interacting with extracellular matrix, other cell adhesion molecules, and the cytoskeleton (Isom et al. 1995; Isom, 2002). The voltage-gated ion channels are encoded by one of the most ancient and conserved gene families, with sequence identity of >50% in the transmembrane domains of human sodium channel α subunits and those of the simplest multicellular eukaryotes. The mammalian genome contains nine functional voltage-gated sodium channel α subunits, which differ in patterns of tissue expression and biophysical properties. The NaV1.1, NaV1.2, NaV1.3 and NaV1.6 channel subtypes, encoded by the SCN1A, SCN2A, SCN3A and SCN8A genes, respectively, are the primary sodium channels in the central nervous system (Catterall, 2000; Goldin et al. 2000; Goldin, 2001; Trimmer & Rhodes, 2004). NaV1.1 and NaV1.3 channels are primarily localized in cell bodies (Westenbroek et al. 1989, 1992), NaV1.2 channels in unmyelinated or pre-myelinated axons and dendrites (Westenbroek et al. 1989, 1992), and NaV1.6 channels in myelinated axons and in dendrites (Caldwell et al. 2000; Krzemien et al. 2000; Jenkins & Bennett, 2001). These channels participate in generation of both somatodendritic and axonal action potentials (Stuart & Sakmann, 1994; Johnston et al. 1996; Callaway & Ross, 1997; Raman & Bean, 1999b; Khaliq & Raman, 2006). In rodents, NaV1.3 channels are highly expressed in the brain during embryonic life, and their expression declines after birth as NaV1.1 and NaV1.2 channels take over (Gordon et al. 1987; Beckh et al. 1989). NaV1.1 expression is first detectable at postnatal day 7 and increases steadily through young adulthood (Gordon et al. 1987; Beckh et al. 1989).
Sodium channels and inherited epilepsy
In spite of their amino acid sequence identity of >70%, knockout of any of the three sodium channel α subunit genes expressed primarily in adult brain (SCN1A, SCN2A and SCN8A) is lethal, demonstrating that each channel performs some non-redundant function. The NaV1.1 channel is remarkable for the number of mutations that cause inherited epilepsy. Screening of human patients with inherited epilepsy first led to the identification of mutations of NaV1.1 channels in two large families with the autosomal dominant epilepsy disorder GEFS+ (generalized epilepsy with febrile seizures plus, OMIM 604233) (Escayg et al. 2000). More than 20 different mutations were subsequently identified in GEFS+ patients, accounting for approximately 10% of cases (Fig. 1). Moreover, a mutation in the NaVβ1 subunit also causes GEFS+ epilepsy, very likely by impairing expression and function of NaV1.1 channels (Wallace et al. 1998). GEFS+ is caused by missense mutations that alter multiple biophysical properties of the channel expressed in non-neuronal cells (Meisler & Kearney, 2005; and see below).
Identification of these familial SCN1A mutations in GEFS+ epilepsy was followed by the surprising report of mutations in children with the sporadic epilepsy disorder SMEI (severe myoclonic epilepsy of infancy or Dravet's syndrome, OMIM 607208) (Claes et al. 2001). These children carry de novo mutations in one allele of the SCN1A gene, leading to haploinsufficiency of NaV1.1 channels (Claes et al. 2001; Ohmori et al. 2002; Sugawara et al. 2002; Claes et al. 2003; Fujiwara et al. 2003; Fukuma et al. 2004). More than 600 SCN1A mutations in the coding sequences of the SCN1A gene have been identified (Fig. 1), accounting for more than 70% of cases (Meisler & Kearney, 2005; http://www.molgen.ua.ac.be/SCN1AMutations/home/Default.cfm). Since only coding regions of the gene are sequenced, it is possible that many of the remaining 30% of SMEI patients harbour mutations in regulatory regions of the gene outside of the coding sequences that impair or prevent channel expression. In addition, duplications and deletions of segments of the SCN1A gene can also impair expression and/or function (Marini et al. 2009). Mutation hotspots, including several sites of CpG deamination, account for approximately 25% of new mutations (Kearney et al. 2006; Depienne et al. 2009). More than half of the SMEI mutations cause loss of function due to stop codons or deletions, demonstrating that haploinsufficiency of SCN1A is pathogenic. Missense mutations of NaV1.1 channels in patients with SMEI are concentrated in the transmembrane segments of the protein, where they may prevent channel expression or severely impair channel function (Fig. 1). In addition, recent studies show that homozygous loss-of-function mutations in the NaVβ1 subunits cause SMEI, probably by impairing expression of NaV1.1 channels on the cell surface (Patino et al. 2009). One practical result of the discovery that haploinsufficiency of NaV1.1 channels causes SMEI is avoidance of treatment with sodium channel blocking anti-epileptic drugs, which exacerbate symptoms in patients with reduced expression of SCN1A (Guerrini et al. 1998; Loscher, 2009).
Severe myoclonic epilepsy of infancy
SMEI begins during the first year of life, with seizures often associated with elevated body temperature due to fever or bathing, and progresses to prolonged, clustered, or continuous seizures and to status epilepticus (Dravet et al. 1992; Engel, 2001). After the second year of life, patients develop co-morbidities including psychomotor delay, ataxia and cognitive impairment. Medically refractory seizures including frequent and prolonged episodes of status epilepticus contribute to an unfavorable long-term outcome (Dravet et al. 1992; Oguni et al. 2001). It is a surprise that haploinsufficiency of a NaV channel causes epilepsy, because reduced sodium current should lead to hypoexcitability rather than hyperexcitability. To understand the mechanistic basis for hyperexcitability and co-morbidities in SMEI, an animal model was generated by targeted deletion or mutation of the Scn1a gene in mouse (Yu et al. 2006).
Selective loss of excitability of GABAergic interneurons and hyperexcitability in SMEI Homozygous null NaV1.1−/− mice developed ataxia and died on postnatal day (P)15, but could be sustained to P17.5 with manual feeding (Yu et al. 2006; Ogiwara et al. 2007). Heterozygous NaV1.1+/− mice exhibited spontaneous seizures and sporadic deaths beginning after P21, with a striking dependence on genetic background (Yu et al. 2006). Loss of NaV1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons (Yu et al. 2006). However, the sodium current density was substantially reduced in inhibitory interneurons of NaV1.1+/− and NaV1.1−/− mice, but not in their excitatory pyramidal neurons (Fig. 2, Table 1). This reduction in sodium current caused a loss of sustained high-frequency firing of action potentials in hippocampal and cortical interneurons (Yu et al. 2006; Ogiwara et al. 2007), thereby impairing their in vivo inhibitory function that depends on generation of high-frequency bursts of action potentials. An immunocytochemical survey also revealed a specific up-regulation of NaV1.3 channels in a subset of hippocampal interneurons, but this up-regulation was insufficient to compensate for the loss of the sodium current of NaV1.1 channels (Yu et al. 2006). These results suggest that reduced sodium currents in GABAergic inhibitory interneurons in NaV1.1+/− heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI. Loss of excitability of GABAergic inhibitory interneurons would allow hyperexcitability of dentate granule and pyramidal neurons, and this gain-of-function effect may cause epilepsy. Failure of firing of additional classes of interneurons in the cerebral cortex and thalamus may also contribute to this complex seizure phenotype.
|Functional effect||Heterozygous knockout||Homozygous knockout|
|Na+ current in hippocampal pyramidal cells (% WT)||100 ± 5.1||96 ± 6.0|
|Na+ current in hippocampal interneurons (% WT)||47.0 ± 7.4||27.5 ± 5.4|
|Na+ current in Purkinje neurons (% WT)|
|Peak||57.6 ± 0.6||41.6 ± 0.5|
|Persistent||44.9 ± 4.1||41.0 ± 3.7|
|Resurgent||49.6 ± 5.5||31.2 ± 3.5|
|Ataxia||Significant at P21||Severe at P11–14|
|Thermally induced seizures||First observed at P20 Increasing thereafter||Not tested|
|Spontaneous seizures||First observed at P21 Increasing thereafter||P11–14|
|Premature death||Increasing premature death after P21||Death at P15|
Loss of excitability of Purkinje neurons and ataxia in SMEI Ataxia, spasticity and failure of motor coordination contribute substantially to the developmental delay and functional impairments of SMEI patients and are major determinants of their poor quality of life, burden of care, and premature deaths (Dravet et al. 2005). How might loss of NaV1.1 channels cause ataxia, spasticity and failure of motor coordination? Purkinje cells are GABAergic inhibitory neurons that serve as the output pathway for information on movement, coordination and balance from the cerebellar cortex. Degeneration of Purkinje neurons and abnormal expression of voltage-gated ion channels in them are associated with ataxia (Fletcher et al. 1996; Raman & Bean, 1997; Grusser-Cornehls & Baurle, 2001; Sausbier et al. 2004). Behavioural assessment indicated severe motor deficits in homozygous NaV1.1 knockout mice, including irregularity of stride length during locomotion, impaired motor reflexes in grasping, and mild tremor in limbs when immobile, consistent with cerebellar dysfunction (Yu et al. 2006; Kalume et al. 2007). A milder impairment of normal gait was observed in the heterozygotes after P21 (Kalume et al. 2007). Immunohistochemical studies showed that NaV1.1 and NaV1.6 channels are the primary sodium channel isoforms expressed in cerebellar Purkinje neurons (Kalume et al. 2007). The amplitudes of whole-cell peak, persistent and resurgent sodium currents in Purkinje neurons were reduced by 58–69%, without detectable change in the kinetics or voltage dependence of channel activation or inactivation (Table 1). Nonlinear loss of sodium current in Purkinje neurons from heterozygous and homozygous mutant animals suggested partial compensatory up-regulation of NaV1.6 channel activity (Table 1). Current-clamp recordings revealed that the firing rates of Purkinje neurons from mutant mice were substantially reduced, with no effect on threshold for action potential generation (Kalume et al. 2007). The results show that NaV1.1 channels play a crucial role in the excitability of cerebellar Purkinje neurons, with major contributions to peak, persistent and resurgent forms of sodium current and to sustained action potential firing. Loss of these channels in Purkinje neurons of mutant mice and SMEI patients may be sufficient to cause their ataxia and related functional deficits. These findings suggest the hypothesis that loss of sodium currents in different classes of GABAergic neurons may underlie the multiple co-morbidities in SMEI, including light hypersensitivity, altered circadian rhythms, and cognitive impairment.
Thermally induced seizures in a mouse model of SMEI Children with SMEI frequently have seizures with elevated body temperature as their first symptom of the disease (Oguni et al. 2005). Experiments with a mouse model of SMEI demonstrated that haploinsufficiency of NaV1.1 channels is sufficient to allow induction of seizures by elevated body temperature (Oakley et al. 2009). P17–18 mice with SMEI did not have thermally induced seizures, but nearly all P20–22 and P30–46 mice with SMEI had myoclonic seizures followed by generalized seizures with elevated core body temperature. Spontaneous seizures were only observed in mice older than P21, indicating that mice with SMEI become susceptible to temperature-induced seizures before spontaneous seizures. Inter-ictal spike activity was seen at normal body temperature in most P30–46 mice with SMEI but not in P20–22 or P17–18 mice, suggesting that inter-ictal epileptic activity correlates with seizure susceptibility. These results define a critical developmental transition for susceptibility to seizures in a mouse model of SMEI and reveal a close correspondence between human and mouse SMEI in the striking temperature and age dependence of SMEI onset and progression.
Balancing excitation and inhibition with genetic compensation and drug treatment The net electrophysiological properties of a neuron are the product of its total ion channel content, so inheritance of genetic variants of ion channels may contribute to polygenic inheritance or variable penetrance among family members. Since SMEI is apparently caused by loss of sodium current and failure of firing of GABAergic interneurons (Yu et al. 2006; Kalume et al. 2007), it may be compensated by mutations that reduce the sodium current and action potential firing of excitatory neurons and thereby re-balance excitation and inhibition in the brain. Such genetic compensation can be studied by mating mouse lines having different well-defined genetic deficiencies. NaV1.6 channels encoded by the Scn8a gene are highly expressed in excitatory neurons, and their functional properties are well suited to driving repetitive firing (Raman & Bean, 1997, 1999a; Chen et al. 2008). Double heterozygous mice with haploinsufficiency for both Scn1a and Scn8a did indeed have reduced susceptibility to drug-induced seizures and improved lifespan compared to NaV1.1 heterozygotes (Martin et al. 2007). These results support the concept that loss-of-function mutations in NaV1.1 channels in SMEI cause an imbalance of excitation over inhibition in the brain and that this imbalance can be partially compensated by a corresponding reduction in the activity of NaV1.6 channels.
In principle, the imbalance of excitation and inhibition can also be corrected by drug treatment. Unfortunately, there are no drugs that selectively inhibit NaV1.6 channels. However, an alternative approach to re-balance excitation and inhibition is to enhance GABAergic neurotransmission by drug treatment. The reduced frequency of action potentials in GABAergic inhibitory neurons in SMEI would decrease phasic release of GABA and impair inhibitory neurotransmission. Drugs such as tiagabine increase the concentration of GABA in the synaptic cleft by inhibiting its reuptake into nerve terminals and glia, and benzodiazepines such as clonazepam increase the response of the postsynaptic GABAA receptors to GABA. Using febrile seizures in a mouse model of SMEI (Oakley et al. 2009) as a test system, we found that combinations of tiagabine and clonazepam are effective in completely preventing thermally induced myoclonic and generalized tonic–clonic seizures (Oakley, Kalume, Scheuer, and Catterall, Dravet Syndrome International Workshop, Abstract, in press, 2010). These encouraging results suggest that similar combination drug therapies may be useful for children with SMEI.
Generalized epilepsy with febrile seizures plus
GEFS+ is usually a much milder epilepsy syndrome than SMEI. Seizures are typically well controlled by treatment with anti-epileptic drugs and no cognitive impairment is observed. The mutations that cause GEFS+ are usually single amino acid missense mutations (Meisler & Kearney, 2005; Fig. 1). Nevertheless, it has been difficult to determine the molecular mechanisms and genotype–phenotype correlations for GEFS+ epilepsy.
Functional effects of GEFS+ mutations expressed in non-neuronal cells Functional effects of GEFS+ mutations were first studied by expression in non-neuronal cells and voltage clamp analysis. The initial study of two mutations inserted in rat NaV1.1 and expressed in Xenopus oocytes revealed that one was a gain-of-function mutation because of destabilized slow inactivation, whereas the second was a loss-of-function mutation because of enhanced slow inactivation (Spampanato et al. 2001). In contrast, the first three mutations inserted in human NaV1.1 and studied by expression in human somatic cells revealed a different functional effect – all three caused impaired inactivation and increased persistent sodium current, leading to the hypothesis that gain-of-function of mutant sodium channels due to loss of inactivation is responsible for GEFS+ epilepsy (Lossin et al. 2002). However, further studies of several GEFS+ mutations expressed in mammalian cells (Lossin et al. 2003; Kahlig et al. 2006) or Xenopus oocytes (Spampanato et al. 2003, 2004; Barela et al. 2006) revealed a mixture of loss-of-function and gain-of-function effects that were caused by several different changes in biophysical properties of NaV1.1 channels (Table 2). Moreover, Rusconi et al. (2007) found that loss-of-function of one GEFS+ mutation resulted from folding and/or trafficking defects that prevented channel expression in the absence of auxiliary β subunits and reduced expression even in the presence of β subunits (Rusconi et al. 2007, 2009). Remarkably, these two GEFS+ mutations can also be partially rescued by treatment with anti-epileptic drugs, which apparently stabilize the mutant channels by binding to them and contributing their binding energy to stabilization of the correctly folded channel protein (Rusconi et al. 2007, 2009). These results indicate that, at least as expressed in non-neuronal cells, mutations that cause GEFS+ epilepsy can have either gain-of-function or loss-of-function effects and these can result from changes in biophysical properties and/or defects in folding and cell surface expression.
|Mutation||Functional effect in vitro||Mechanism||Functional effect in vivo||Mechanism||Reference|
|D188V||GoF||Impaired slow inactivation||Cossette et al. (2003)|
|R859C||LoF||Positive shift of activation; slowed recovery from slowed inactivation; reduced INa*||Barela et al. (2006)|
|T875M||LoF||Enhanced slow inactivation||Spamanato et al. (2001)|
|Increased persistent INa**||Lossin et al. (2002)|
|W1204R||GoF||Increased persistent INa**||Lossin et al. (2002)|
|GoF||Negative shift of activation and inactivation; negative shift of window current*||Spampanato et al. (2003)|
|R1648H||GoF||Enhanced recovery from inactivation*;|| Spampanato et al. (2001) |
Lossin et al. (2002)
|Increased persistent INa**||Tang et al. (2009)|
|LoF||Increased inactivation; impaired recovery from inactivation||Martin et al. (2010)|
|I1656M||LoF||Positive shift of activation||Lossin et al. (2003)|
|R1657C||LoF||Positive shift of activation; reduced expression|| Lossin et al. (2003) |
Vanoye et al. (2005)
|M1841T||LoF||Folding/trafficking defect**||Rusconi et al. (2007)|
|D1866Y||GoF||Impairment of the effect of beta subunits to enhance inactivation*||Spampanato et al. (2004)|
|R1916G||LoF||Folding/trafficking defect**||Rusconi et al. (2009)|
GEFS+ mutations in mouse genetic models Considering the confusing picture from studies of the functional effects of GEFS+ mutations in transfected non-neuronal cells, it is important to determine the functional effects of these mutations in neurons in vivo. Toward this end, the mutation R1648H was incorporated into the mouse genome using a Bac transgene strategy, providing an animal model of GEFS+ that permits more detailed analysis of the effect of mutations on neuronal sodium currents in vivo. The transfected NaV1.1 channel contained both the GEFS+ mutation and an additional amino acid substitution that prevents block by the pore-blocker tetrodotoxin (TTX), thereby allowing selective block of endogenous sodium channels by TTX without effect on the transfected NaV1.1 channels (Tang et al. 2009). Mice expressing the transgene had increased sodium channel expression and reduced threshold for seizure induction by kainic acid. The level of transgene-induced sodium current was much greater in inhibitory neurons than in excitatory neurons, as expected from previous studies of NaV1.1 knockout mice showing selective expression in inhibitory neurons (Yu et al. 2006; Ogiwara et al. 2007). The R1648H channels showed reduced function in both excitatory and inhibitory neurons, but the biophysical mechanisms were different – reduced peak sodium currents and enhanced slow inactivation in inhibitory neurons versus negatively shifted voltage dependence of fast inactivation in excitatory neurons. These functional effects were predicted to have the net result of reduced excitability of inhibitory neurons. Thus, this GEFS+ mutation causes selective impairment of excitability of GABAergic inhibitory neurons in vivo (Tang et al. 2009), as previously demonstrated for SMEI mutations (Yu et al. 2006). More recent studies of a mouse model in which the R1648H mutation has been inserted into the mouse genome under the native promoter lead to similar conclusions (Martin et al. 2010). In light of these results, GEFS+ and SMEI may be caused by a continuum of mutational effects that selectively impair firing of GABAergic inhibitory neurons. According to this hypothesis, mild impairment of NaV1.1 channels and action potential firing of GABAergic neurons causes GEFS+ epilepsy, whereas complete (or nearly complete) loss of NaV1.1 channel function causes more severe impairment of action potential firing of GABAergic neurons and leads to SMEI.
Potential role of mutations in NaV1.1 channels in febrile seizures in childhood
Febrile seizures are common in childhood, but the basis for their prevalence is not known. Several lines of evidence now suggest that mild loss-of-function mutations or polymorphisms in NaV1.1 channels may cause a significant portion of febrile seizures. Mantegazza et al. (2005) characterized a mild loss-of-function mutation in NaV1.1 channels in a family with familial febrile seizures. This mutation caused reduction of peak sodium currents and positive shift in the voltage dependence of activation when expressed in non-neuronal cells. Therefore, this study provided the first evidence for association of mild loss of function of NaV1.1 channels with familial febrile seizures.
There has been considerable controversy regarding claims that routine childhood vaccination may be associated with the onset of both febrile and afebrile seizures and mental decline. Berkovic et al. (2006) studied 14 children with this diagnosis and identified SCN1A mutations in 11 of the children. Their observations indicate that vaccination and its associated fever may trigger the first seizure episode of an underlying genetic disorder, GEFS+ or SMEI, which often presents first as febrile seizures. These studies further implicate dysfunction of NaV1.1 channels in childhood febrile seizures.
Most recently, human genetic studies have also suggested an association of genetic alterations in NaV1.1 channels with non-familial febrile seizures (Schlachter et al. 2009). In normal development, the mRNAs encoding NaV1.1, NaV1.2 and NaV1.3 channels undergo a regulated change in alternative splicing of exon 5 (Sarao et al. 1991; Gazina et al. 2009), which has a striking effect on the voltage dependence of channel activation (Auld et al. 1990). Regulation of this alternative splicing process is disrupted by a single nucleotide polymorphism (SNP IVS5N+5 G>A; Tate et al. 2005, 2006). The presence of this SNP has been correlated with altered response to anti-epileptic drugs (Tate et al. 2005, 2006) and with risk of febrile seizures in a large cohort of epilepsy patients from Germany and Austria (Schlachter et al. 2009). On the other hand, correlation of this SNP with febrile seizures was not observed in a similar study of Australian epilepsy patients (Petrovski et al. 2009). Nevertheless, even considering these negative data, the combination of results from studies of familial febrile seizures (Mantegazza et al. 2005), vaccination-related seizures (Berkovic et al. 2006), responsiveness to anti-epileptic drugs (Tate et al. 2005, 2006), and febrile seizures in a German/Austrian cohort of patients (Schlachter et al. 2009) all point to a key role of NaV1.1 channels as molecular determinants of the risk of epilepsy in the general population and raise the possibility that a significant fraction of febrile seizures in children are caused by mild loss-of-function mutations or polymorphisms of NaV1.1 channels in combination with environmental precipitating factors.
A unified loss-of-function hypothesis for NaV1.1 genetic epilepsies
NaV1.1 channels are highly expressed in many GABAergic inhibitory neurons and are responsible for essentially all of the sodium current in the cell bodies of hippocampal interneurons (Yu et al. 2006). Loss of function of these sodium channels greatly impairs the ability of these inhibitory neurons to fire action potentials at high frequency and therefore would greatly reduce their phasic release of GABA (Yu et al. 2006). It is likely that this loss of action potential firing by inhibitory neurons leads to an imbalance of excitation and inhibition in the brain and consequently to febrile seizures and epilepsy. In embryonic and neonatal rodent brain, intracellular Cl− concentration is high, and activation of GABA-A receptors can depolarize neurons by conducting Cl− outward (Rivera et al. 1999; Blaesse et al. 2009). However, by postnatal day 22, when spontaneous seizures begin in mouse models of NaV1.1 epilepsies, intracellular concentration of Cl− is reduced owing to increased expression and function of cation–hloride co-transporters, and Cl− conductance resulting from activation of GABA-A receptors hyperpolarizes neurons and clamps the membrane potential near its resting level (Rivera et al. 1999; Blaesse et al. 2009).
Although more work is needed to develop definite genotype–phenotype correlations for the NaV1.1 epilepsies, we extend a previous proposal (Ragsdale, 2008) and propose the unifying hypothesis that the spectrum of severity of the NaV1.1-associated forms of epilepsy results from a spectrum of increasing severity of loss-of-function mutations of NaV1.1 channels and increasing impairment of action potential firing in GABAergic inhibitory neurons (Fig. 3). Mild impairment of NaV1.1 channel function causes febrile seizures; moderate to severe impairment of NaV1.1 function by missense mutations and/or altered mRNA processing causes the range of phenotypes observed in GEFS+ epilepsy; and very severe to complete loss of function causes SMEI. The severity of phenotype in these genetic diseases is also influenced strongly by genetic background effects as illustrated by striking differences in phenotypes among GEFS+ patients with the same missense mutation (Scheffer et al. 2009), different severity of disease of SMEI patients with complete loss-of-function mutations (Mulley et al. 2005; Harkin et al. 2007), and dramatic differences in sensitivity among mouse strains to the same loss-of-function mutations (Yu et al. 2006). We hope that this unifying hypothesis and further analysis of the effects of mutations in mouse models will bring increasing clarity to our understanding of genotype-phenotype correlations in this family of epilepsy syndromes and will provide fresh insights into effective therapies.