Epilepsy Research 54 (2003) 201–207

Nav1.1 channels with mutations of severe myoclonicepilepsy in infancy display attenuated currents

Takashi Sugawaraa, Yuji Tsurubuchib, Tateki Fujiwarac,Emi Mazaki-Miyazakia, Keiichi Nagatab, Mauricio Montald,

Yushi Inouec, Kazuhiro Yamakawaa,∗a Laboratory for Neurogenetics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

b Laboratory for Memory and Learning, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japanc National Epilepsy Center, Shizuoka Medical Institute of Neurological Disorders, 886 Urushiyama, Shizuoka 420-8688, Japan

d Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA

Received 28 January 2003; received in revised form 10 April 2003; accepted 14 April 2003

Abstract

Severe myoclonic epilepsy in infancy (SMEI) is characterized by intractable febrile and afebrile seizures, severe mentaldecline, and onset during the first year of life. Nonsense, frameshift, and missense mutations of SCN1A gene encoding thevoltage-gated Na+ channel�-subunit type I (Nav1.1) have been identified in patients with SMEI. Here, we performed whole-cellpatch-clamp analyses on HEK293 cells expressing human Nav1.1 channels bearing SMEI nonsense and missense mutations. Themutant channels showed remarkably attenuated or barely detectable inward sodium currents. Our findings indicate that SMEImutations lead to loss-of-function and may contribute to the development of SMEI phenotypes.© 2003 Elsevier Science B.V. All rights reserved.

Keywords: Severe myoclonic epilepsy in infancy; SMEI; Mutations; Sodium channel; SCN1A; Patch-clamp

1. Introduction

Severe myoclonic epilepsy in infancy (SMEI)(Dravet, 1978; Dravet et al., 1982) is classified as“syndrome undetermined as to whether it is focal orgeneralized,” and characterized by various signs in-

Abbreviations: SMEI, severe myoclonic epilepsy in infancy;GEFS+, generalized epilepsy with febrile seizures plus

∗ Corresponding author. Tel.:+81-48-467-9703;fax: +81-48-467-7095.

E-mail address: yamakawa@brain.riken.go.jp (K. Yamakawa).URL: http://www.brain.riken.go.jp/labs/ngs/index.html.

cluding conspicuous fever-sensitive generalized tonic-clonic, alternative hemiclonic, myoclonic, atypicalabsence, complex partial seizures, and photosensi-tivity (Commission on Classification and Termino-logy of the International League Against Epilepsy,1989). SMEI has an onset during the first year of lifeand is followed by severe mental decline. Frequentseizures are often prolonged and resistant to mostanti-epileptic drugs (Oguni et al., 2001).

Recently, mutations of SCN1A, the gene encodingvoltage-gated Na+ channel�-subunit type I (Nav1.1),were reported in SMEI patients (Claes et al., 2001).

0920-1211/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0920-1211(03)00084-6

202 T. Sugawara et al. / Epilepsy Research 54 (2003) 201–207

Mutations were identified in all seven cases studied:four frameshift, one nonsense, one splice-donor, onemissense, and all were heterozygous and de novomutations. Subsequently, we reported frequent trunca-tion mutations of SCN1A in Japanese SMEI patients(Sugawara et al., 2002). Nonsense or frameshift mu-tations resulting in truncated channels are a majorcomponent in typical SMEI patients, however recentlywe and other groups reported that missense mutationsalso play roles in the SMEI phenotype (Ohmori et al.,2002; Fujiwara et al., 2003). Mutations of SCN1Ahave also been known to be responsible for general-ized epilepsy with febrile seizures plus (GEFS+) thatis a milder idiopathic epilepsy (Escayg et al., 2000,2001; Wallace et al., 2001; Sugawara et al., 2001a).A term autosomal dominant epilepsy with febrileseizures plus (ADEFS+) has also been proposed in-stead of GEFS+ because of the occasional associationof partial seizures (Ito et al., 2002). In contrast to theSMEI truncation mutations, the GEFS+ mutationsare exclusively missense type.

Nav1.1 has four homologous repeats (DI–DIV) eachconsisting of six transmembrane helices (S1–S6). Poreregion (S5, S6 and loop between them), voltage sen-sor (S4), phosphorylation sites (intracellular loop), andinactivation gate (DIII–DIV loop) have been identi-fied and characterized by the site-directed mutagenesis(Catterall, 2000). Nav1.1 is primarily expressed at thesoma of neuronal cells in the CNS (Gong et al., 1999)and is responsible for the rising phase of the actionpotential. The functional effects of GEFS+ missensemutations of SCN1A have been investigated by threegroups (Alekov et al., 2001; Spampanato et al., 2001;Lossin et al., 2002). Biophysical characterization ofthe mutant channels has revealed alterations of the in-activation process as a major factor contributing topersistent Na+ influx.

GEFS+ and SMEI are caused by gene deficits inthe same gene, whereas there are many differencesbetween their phenotypes (Scheffer et al., 2001; Singhet al., 2001). Functional analyses of channel proteinswith those mutations may elucidate the phenotypicdiversity. Here we present a biophysical study ofchannels with six SMEI-associated mutations. Bothnonsense and missense SCN1A mutations resulted inloss-of-function and lead to a reduction of the sodiumcurrent which may contribute to the pathogenesis ofSMEI.

2. Experimental

2.1. SMEI mutations

Clinical data of patients with SMEI harboring muta-tions studied here were reported elsewhere (Fujiwaraet al., 2003). All patients but one (G979R) met thecriteria of SMEI according to the proposal of theCommission on Classification and Terminology of theInternational League Against Epilepsy (1989). The pa-tient with G979R mutation did not have a history ofmyoclonic seizures but all other clinical features aresame as SMEI (Fujiwara et al., 2003).

2.2. Molecular cloning of human SCN1A cDNAand mutagenesis

5′- and 3′-RACE reactions were carried out withMarathon-ReadyTM cDNA kit (Clontech, Palo Alto,CA) to obtain human voltage-gated sodium channel�Igene (SCN1A) cDNA. Two human adult brain cDNAlibraries (Clontech) was screened by hybridizationwith the RACE products as probes to obtain morethan 10 cDNA clones showed a consensus sequence(Genbank accession no. AY043484) that is shorterby 33 nucleotides (c.2111–2143: corresponding to 11amino acids) than reported full-length cDNA (Gen-bank accession no. NM006920:Escayg et al., 2000;Lossin et al., 2002). For the purpose of conveniences,the amino acid numberings are coincided to Escayg’ssequence. The cDNA harboring full-length ORF butlacking the 33 nucleotides was inserted into NotIand ApaI sites of pcDNA3.1MycHisB(+) plasmid(Stratagene, La Jolla, CA), and mutant Na+ chan-nels harboring mutations R712X, G979R, N985I,R1407X, F1831S, and R1892X, were prepared usinga site-directed mutagenesis kit (QuikChange, Strata-gene). Mutated sites as well as full insert sequencesfor all clones were confirmed by direct sequencing.

2.3. Transfection and Western Blotting

Plasmids bearing wild-type (WT) and mutant�-subunit cDNA as well as 1/10 w/w pEGFPC2 vector(Clontech) as a marker were transiently co-transfectedinto HEK293 human embryonic kidney cells byLipofectamine 2000 (Life Technologies, Rockville,MD) as recommended by the manufacturer. Transient

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expression of functional Na+ channels was detectedafter 20–36 h.

Transfected cells were lysed by addition of 100 mlof hot (80◦C) 1 × SDS loading buffer. Sampleswere boiled, sonicated and centrifuged at 3000× g

for 5 min at 4◦C to remove insoluble material andthe supernatant was used as total lysate. Super-natants were collected and run on 2–15% gradientgels, and then, transferred onto nitrocellulose filter(0.45�m, Schleicher & Schuell, Dassel, Germany).After blocking, membranes were processed throughsequential incubations with anti-Nav1.1 antibody(Alomone Labs, Jerusalem, Israel) for 1 h, and thenwith 0.4�g/ml horseradish peroxidase-conjugatedanti-rabbit IgG (Santa Cruz Biotechnology, SantaCruz, CA). Immunoreactive proteins on the filterwere visualized using the Renaissance Chemilumi-nescence kit (NEN Life Science Products, Boston,MA).

2.4. Patch-clamp analysis

Cells were plated on poly-l-lysine-coated glass cov-erslips (Biocoat Cellware Poly-l-lysine 12 mm Cov-erslip, Becton Dickinson Labware, Bedford, MA) andmaintained in culturing medium (Dulbecco’s Modi-fied Eagle’s Medium supplemented with 10% fetalbovine serum) before patch-clamp experiments in anenvironment of humidified 5% CO2.

Membrane currents were recorded using thewhole-cell configuration of the patch-clamp techniqueusing an Axopatch 200B amplifier (Axon Instru-ments, Burlingame, CA) at room temperature (22◦C)as previously described (Sugawara et al., 2001b).Signals were stored on a hard disk in a MacintoshG3/266 (Apple Computer Inc., Cupertino, CA) usingPulse+PulseFit 8.11 (HEKA, Lambrecht, Germany).Capacitative and leakage currents were digitally sub-tracted by using the P+P/4 procedure. The peakNa+ current arising from endogenous Na+ channelspresent in HEK cells was less than 50 pA. Na+ cur-rents for mutant channels were recorded from at leastfive GFP-labeled cells.

Conductance–voltage relationship: Currents wereevoked by 10 ms depolarizations to various levelsfrom a holding potential of−120 mV. The conduc-tance (gNa) was calculated according to the equation,gNa = INa/(Vg − Vr), (INa is the peak amplitude

of Na+ current, Vg is the test potential, andVr isthe reversal potential for Na+). The curves weredrawn according to the equation,gNa/maxgNa =1/{1+ exp[Vg1/2 − Vg]/kg}, (maxgNa is the maximumvalue for gNa, Vg1/2 is the half maximal potential ofmaxgNa, andkg is the slope factor).

Steady-state inactivation: The membrane potentialwas held at various levels for 2 s, and then Na+ cur-rent was evoked by a step depolarization to−10 mV.The curves were drawn according to the Boltzmannequation,I/Imax = {1 + exp[(Vh − V1/2)/k]}−1, (Vhis the holding potential,V1/2 is the half maximal in-activation, andk is the slope factor).

The internal pipette solution contained (in mM):CsF 135, NaCl 10, and HEPES-acid 5. The pHwas adjusted to 7.0 with CsOH, and the osmolar-ity was 276 mOsm. The usage of fluoride is criticalto obtain stable currents. The external solution con-tained (in mM): NaCl 135, CaCl2 2.0, MgCl2 1.0,HEPES-acid 10.0, and glucose 5.0. The solution wasadjusted to pH 7.4 with NaOH and the osmolarity was281 mOsm.

3. Results

The gating kinetics of channels with SMEI-asso-ciated nonsense mutations of SCN1A (R712X,R1407X, and R1892X) (Sugawara et al., 2001a) andmissense mutations (G979R, N985I, and F1831S)(Fujiwara et al., 2003) were analyzed. The loca-tions of these mutations are illustrated inFig. 1.All mutant channels were transiently expressed inHEK293 cells, and expression levels and molecularsizes of the transcripts for the WT and all mutantchannels were assessed by immuno-blot analysis(data not shown). Although all mutant channels wereexpressed in HEK cells, patch-clamp analyses offour mutant channels (G979R, N985I, R712X, andR1407X) disclosed extremely small Na+ currents(Fig. 2A, B, E, and F). The maximal evoked currentswere marginally larger than the endogenous Na+currents (<50 pA) present in HEK cells. The mis-sense F1831S mutant channel exhibited a reductionof the peak Na+ current (Fig. 2C), and the nonsenseR1892X mutant channel showed a drastic attenuationof the peak Na+ current compared to the WT channel(Fig. 2G).

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Fig. 1. Locations of SMEI-associated missense and nonsense mutations on SCN1A neuronal sodium channel�-subunit type-1 gene. The�-subunit consists of four homologous domains (D1–D4), and each domain consists of six transmembrane segments (S1–S6). G979R andN985I are located at D2S6 that is predicted to face the pore lumen. F1831S is located at C-terminus region.�: Nonsense mutation.�:Missense mutation.

4. Discussion

The biophysical analysis of human Nav1.1 channelsbearing SMEI-associated nonsense and missense mu-tations described here indicates that such mutationswould lead to loss-of-function with a profound decre-ment or absence of Na+ currents. It is conceivablethat the truncated channels with R712X and R1407Xmutations generate non-functional channels giventhe large missing domains (Fig. 1). By contrast, thechannel with the R1892X mutation produced smallyet detectable sodium currents, presumably becauseit allows expression of a protein retaining the fourhomologous repeats intact and a small yet significantsegment of the C-terminus. G979R and N985I mis-sense mutations are located at the critical D2S6 re-gion that faces the inner pore, and may be critical forchannel function. The remaining missense mutationF1831S is located at the C-terminus, again empha-sizing a critical role of the C-terminal cytoplasmicregion for maintaining the intact function of sodiumchannel �-subunits as shown for SCN5A (Rivoltaet al., 2001; Cormier et al., 2002).

The functional deficits of channels with SMEImutations appear to be opposite to those exhibitedby channels with GEFS+ mutations, which mostlylead to gain-of-function. GEFS+ missense mutationsof SCN1A (Alekov et al., 2001; Spampanato et al.,2001; Lossin et al., 2002), SCN2A (Sugawara et al.,2001b), and SCN1B (Wallace et al., 1998; Meadows

et al., 2002) affect the gating kinetics and slow in-activation. One group reported opposite effects fortwo different GFES+ missense mutations of SCN1Aleading either to augment or reduce sodium current,and suggested that either increase or decrease ofsodium ion influx may cause the GFES+ phenotypes(Spampanato et al., 2001). However, a recent studyon the same GEFS+ mutations of SCN1A showedthat those mutations produced persistent inwardcurrents, which were proposed to underlie the neu-ronal hyperexcitability and lead to epileptic seizures(Lossin et al., 2002). This is in agreement with ourprevious study on the SCN2A missense mutationassociated with GFES+ (Sugawara et al., 2001b).Even though SMEI and GEFS+ share overlappingphenotypes such as febrile generalized tonic-clonicseizures and various afebrile seizures, symptoms aredistinct at the point of seizure frequency, therapy re-sistance and mental decline (Benlounis et al., 2001).At this moment, it would be a plausible explanationthat SCN1A mutations leading to gain-of-function,increase of sodium currents, cause GEFS+ andthose leading loss-of-function, decrease of currents,cause SMEI.

There has been much discussion on the subject ofdysfunctional voltage-gated sodium channels. A casein point is the cardiac voltage-gated sodium channel(SCN5A) for which frameshift and missense muta-tions have been implicated in the long QT syndrometype 3 (LQT3) (Wang et al., 1995) and the Brugada

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Fig. 2. Representative whole-cell current recordings in HEK293 cells expressing WT human Nav1.1 channel, and those with SMEI-associatedmutations. Currents were evoked from a holding potential of−120 to 0 mV. More than 10 fluorescent-active cells were recorded for eachmutant channel, and maximal sodium currents were shown in the figure. (A–C): Mutant channels bearing missense mutations (G979R,N985I, and F1831S). (D): WT control. (E–G): Mutant channels bearing nonsense mutations (R712X, R1407X, and R1892X).

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syndrome (BrS) (Chen et al., 1998). Although thesesyndromes show clinical overlaps, such as nocturnalsudden cardiac death without prior symptoms, theelectrocardiogram (EKG) features are distinct; fur-thermore patients with BrS display a more severephenotype which is rather resistant to treatments.Individual identical mutations of SCN5A could leadto both BrS and LQT3 (Bezzina et al., 1999), butmost of the LQT3 mutations are missense, some ofthose revealed gained function, such as slowed inac-tivation leading to increase of currents, whereas forBrS the mutations encompass missense, nonsense,and frameshift which lead to non-functional channels.These observations are consistent with the findings onSCN1A mutations in patients with GEFS+ and SMEI.

Here we demonstrated that channels bearing SMEImutations displayed attenuated Na+ currents, and pro-posed that such loss-of-function may contribute tothe SMEI phenotypes. Studies on further cases, how-ever, would be necessary to understand the molecularpathology of SMEI.

Acknowledgements

We are thankful to Dr. Makoto Kaneda (Depart-ment of Physiology, Keio University) for his helpfulcomment. This study was supported in part by grantsfrom the Ministry of Education, Science and Cultureof Japan. Research at the University of California issupported by NIH Grant GM-49711.

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