Functional characterization of the D188V mutation in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS)
Introduction
Epilepsy is a common neurological disorder, affecting approximately 1% of the population. Recently, genes underlying some of the idiopathic partial and generalized epileptic syndromes have been identified. The vast majority of the genes predisposing to familial epilepsy in humans incriminate ion channels. Hence, mutations have been found in voltage-gated sodium and potassium channels (SCN1B, SCN1A, KCNQ2, KCNQ3) (Wallace et al., 1998, Escayg et al., 2000, Charlier et al., 1998, Biervert et al., 1998, Singh et al., 1998), as well as in ligand-gated channels (GABRA1, GABRG2, CHRNA4, CHRNB2) (Cossette et al., 2002, Baulac et al., 2001, Steinlein et al., 1995, De Fusco et al., 2000). Among these epilepsy genes, mutations in the alpha 1 subunit of the voltage-gated sodium channel (SCN1A) are the most frequently encountered, and have been found to cause at least three different epileptic syndromes: generalized epilepsy associated with febrile seizure (GEFS) (Escayg et al., 2000), febrile seizures associated with temporal lobe epilepsy (Sugawara et al., 2001a) and severe myoclonic epilepsy of infancy (Claes et al., 2001). However, even if SCN1A is increasingly recognized as an important gene causing epilepsy in humans, the mechanism by which these mutations in SCN1A lead to spontaneous and uncontrolled discharges of neuronal cells remains elusive. Several recent studies have assessed the functional effect of mutations in SCN1A, focusing on three mutations identified in families segregating GEFS (T875M, W1204R and R1648H) (Alekov et al., 2000, Alekov et al., 2001, Spampanato et al., 2001, Lossin et al., 2002). These functional studies suggest that sodium channel mutations contribute to neuronal hyperexcitability through subtle changes in persistent sodium current (Lossin et al., 2002), activation, inactivation or recovery from inactivation (Alekov et al., 2000, Alekov et al., 2001, Spampanato et al., 2001). In this study, we present the identification of a D188V mutation in SCN1A in a large family segregating GEFS that we have previously mapped to chromosome 2q23-24 (Lopes-Cendes et al., 2000). This mutation is located in the intracellular loop very close to the S3 transmembrane segment of domain I, which has no previously identified role in voltage-gating. Electrophysiological analysis of D188V mutant channels expressed in HEK cells shows that the mutation causes decreased cumulative inactivation of sodium current during high frequency channel activity, an effect compatible with membrane hyperexcitability. This mechanism could be central in the pathophysiology of the epilepsies caused by mutations in sodium channels in humans.
Section snippets
Screening for mutation
Family collection and phenotypes have been described previously (Lopes-Cendes et al., 2000). The genomic organization of the human SCN1A gene was determined by partial sequencing of human PAC clones isolated from a chromosome 2 enriched PAC sublibrary (Gingrich et al., 1996) probed at low stringency with a rat SCN2A cDNA (Auld et al., 1988). Primers were designed to amplify 200–350 bp fragments from genomic DNA to screen all coding portions of the gene.
Portions of the SCN1A gene were amplified
Mutation screening
To identify mutation in our GEFS family, we initially performed SSCP analysis on three affected individuals from the pedigree, and on three normal controls using primers specific to the human SCN1A gene. A SSCP variant was detected in exon 4 with primers NaC-63 and NaC-64 for all of the affected patients, and none of the controls. No mutation was found in the other exons of the SCN1A gene. Sequence analysis determined that the patient was heterozygous for an A-T substitution predicted to change
Discussion
In the present study, we describe a D188V mutation in the SCN1A gene, in a family that we have previously linked to chromosome 2p36 (GEFS2) (Lopes-Cendes et al., 2000). Several lines of evidence suggest that D188V is the pathogenic mutation in this family: (1) the non-conservative change of an aspartic acid (charged polar) to a valine (uncharged, nonpolar); (2) D188 is conserved in ten different sodium channel alpha subunits found in humans, and also in different sodium channels from
Acknowledgements
The authors wish to thank E. Schurr for chromosome specific PAC clones. P.C. is funded by a Canadian Institutes for Health Research (CIHR) fellowship, clinical-scientist program (FRN-35318). D.S.R. is funded by CIHR grant MT-13485. G.A.R. is supported by the CIHR. This work was supported by Xenon Genetics Inc.
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2010, Biochemical and Biophysical Research CommunicationsCitation Excerpt :All these animals exhibited a marked reduction in sodium currents in inhibitory GABAergic interneurons, suggesting that the truncated mutations of Scn1a inhibit GABAergic activity to confer the seizure vulnerability. Nonetheless, it is known that the missense mutations of Scn1a gene reported in FS patients produce a variety changes in the channel functions such as gain-of-function changes (e.g., increased persistent leak current, depolarized shift in voltage-dependence of inactivation, hyperpolarized shift in voltage-dependence of activation), loss-of-function changes (e.g., hyperpolarized shift in voltage-dependence of inactivation and depolarized shift in voltage-dependence of activation) and mixed patterns of the above changes [6,8,21–25]. Due to the diversity of these functional alterations, influences of the Scn1a missense mutations on the GABAergic neural network or synaptic transmission remain to be clarified.
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These authors contributed equally to this work.