Nav1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents
Introduction
Severe myoclonic epilepsy in infancy (SMEI) (Dravet, 1978, Dravet et al., 1982) is classified as “syndrome undetermined as to whether it is focal or generalized,” and characterized by various signs including conspicuous fever-sensitive generalized tonic-clonic, alternative hemiclonic, myoclonic, atypical absence, complex partial seizures, and photosensitivity (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). SMEI has an onset during the first year of life and is followed by severe mental decline. Frequent seizures are often prolonged and resistant to most anti-epileptic drugs (Oguni et al., 2001).
Recently, mutations of SCN1A, the gene encoding voltage-gated Na+ channel α-subunit type I (Nav1.1), were reported in SMEI patients (Claes et al., 2001). Mutations were identified in all seven cases studied: four frameshift, one nonsense, one splice-donor, one missense, and all were heterozygous and de novo mutations. Subsequently, we reported frequent truncation mutations of SCN1A in Japanese SMEI patients (Sugawara et al., 2002). Nonsense or frameshift mutations resulting in truncated channels are a major component in typical SMEI patients, however recently we and other groups reported that missense mutations also play roles in the SMEI phenotype (Ohmori et al., 2002, Fujiwara et al., 2003). Mutations of SCN1A have also been known to be responsible for generalized epilepsy with febrile seizures plus (GEFS+) that is a milder idiopathic epilepsy (Escayg et al., 2000, Escayg et al., 2001, Wallace et al., 2001, Sugawara et al., 2001a). A term autosomal dominant epilepsy with febrile seizures plus (ADEFS+) has also been proposed instead of GEFS+ because of the occasional association of partial seizures (Ito et al., 2002). In contrast to the SMEI truncation mutations, the GEFS+ mutations are exclusively missense type.
Nav1.1 has four homologous repeats (DI–DIV) each consisting of six transmembrane helices (S1–S6). Pore region (S5, S6 and loop between them), voltage sensor (S4), phosphorylation sites (intracellular loop), and inactivation gate (DIII–DIV loop) have been identified and characterized by the site-directed mutagenesis (Catterall, 2000). Nav1.1 is primarily expressed at the soma of neuronal cells in the CNS (Gong et al., 1999) and is responsible for the rising phase of the action potential. The functional effects of GEFS+ missense mutations of SCN1A have been investigated by three groups (Alekov et al., 2001, Spampanato et al., 2001, Lossin et al., 2002). Biophysical characterization of the mutant channels has revealed alterations of the inactivation process as a major factor contributing to persistent Na+ influx.
GEFS+ and SMEI are caused by gene deficits in the same gene, whereas there are many differences between their phenotypes (Scheffer et al., 2001, Singh et al., 2001). Functional analyses of channel proteins with those mutations may elucidate the phenotypic diversity. Here we present a biophysical study of channels with six SMEI-associated mutations. Both nonsense and missense SCN1A mutations resulted in loss-of-function and lead to a reduction of the sodium current which may contribute to the pathogenesis of SMEI.
Section snippets
SMEI mutations
Clinical data of patients with SMEI harboring mutations studied here were reported elsewhere (Fujiwara et al., 2003). All patients but one (G979R) met the criteria of SMEI according to the proposal of the Commission on Classification and Terminology of the International League Against Epilepsy (1989). The patient with G979R mutation did not have a history of myoclonic seizures but all other clinical features are same as SMEI (Fujiwara et al., 2003).
Molecular cloning of human SCN1A cDNA and mutagenesis
5′- and 3′-RACE reactions were carried out
Results
The gating kinetics of channels with SMEI-associated nonsense mutations of SCN1A (R712X, R1407X, and R1892X) (Sugawara et al., 2001a) and missense mutations (G979R, N985I, and F1831S) (Fujiwara et al., 2003) were analyzed. The locations of these mutations are illustrated in Fig. 1. All mutant channels were transiently expressed in HEK293 cells, and expression levels and molecular sizes of the transcripts for the WT and all mutant channels were assessed by immuno-blot analysis (data not shown).
Discussion
The biophysical analysis of human Nav1.1 channels bearing SMEI-associated nonsense and missense mutations described here indicates that such mutations would lead to loss-of-function with a profound decrement or absence of Na+ currents. It is conceivable that the truncated channels with R712X and R1407X mutations generate non-functional channels given the large missing domains (Fig. 1). By contrast, the channel with the R1892X mutation produced small yet detectable sodium currents, presumably
Acknowledgements
We are thankful to Dr. Makoto Kaneda (Department of Physiology, Keio University) for his helpful comment. This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan. Research at the University of California is supported by NIH Grant GM-49711.
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