A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

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Abstract

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

Introduction

Voltage-gated sodium channels (VGSCs) play a critical role in the regulation of neuronal excitability by facilitating the initiation and propagation of action potentials. VGSCs consist of a pore-forming, 260-kD α subunit (Nav1.1 to Nav1.9) that is associated with two of four accessory β subunits (β1 to β4) in the central nervous system (CNS). The α subunits are responsible for voltage sensing and ion conductance and the β subunits modulate channel kinetics and membrane localization.

Mutations in the SCN1A gene encoding Nav1.1 have been established as causing several subtypes of epilepsy, including Generalized Epilepsy with Febrile Seizures Plus (GEFS+, MIM 604233) and Severe Myoclonic Epilepsy of Infancy (SMEI or Dravet Syndrome, MIM 607208) (Escayg et al., 2000, Escayg et al., 2001, Claes et al., 2001, Ohmori et al., 2003, Fujiwara et al., 2003, Wallace et al., 2001, Wallace et al., 2003, Sugawara et al., 2001, Nagao et al., 2005, Mantegazza et al., 2005, Annesi et al., 2003). GEFS+ is a familial disorder characterized by febrile seizures that persist beyond the age of six, with a wide range of afebrile seizure types and severities among affected family members. SMEI is an intractable childhood epilepsy disorder that is often associated with mental retardation and ataxia. Although great strides have been made towards identifying the genetic basis for these disorders, the mechanisms that lead to seizure generation remain poorly understood.

The SCN1A mutation R1648H was first identified in a large GEFS+ family (Escayg et al., 2000). The invariant, positively charged R1648 residue is located in the voltage-sensing S4 segment of the fourth homologous domain (D4) of the α subunit. Previous electrophysiological analyses of the R1648H mutation identified different alterations in sodium channel gating kinetics, namely accelerated recovery from inactivation and decreased use-dependent inactivation of channel activity (Spampanato et al., 2001), as well as increased persistent inward current during sustained depolarization (Lossin et al., 2002, Vanoye et al., 2006). These differences could be due to the fact that these experiments used different non-neuronal heterologous expression systems — Xenopus oocytes (Spampanato et al., 2001) and tsA201 cells (Lossin et al., 2002), a derivative of HEK293 human embryonic kidney cells. Neither system is a good model of a neuron. Mutation of R1648 to cysteine causes SMEI (Ohmori et al., 2003) and results in significant impairment of fast inactivation, including persistent, noninactivating channel activity and a depolarized shift in the voltage dependence of activation (Rhodes et al., 2004).

To study R1648H mutant Nav1.1 channels in their native neuronal environment, we generated a mouse model of GEFS+. We used a bacterial artificial chromosome (BAC) transgenic strategy in which the R1648H mutation was introduced into the orthologous position of the mouse Scn1a gene contained in a BAC clone. The BAC was also modified to carry a FLAG epitope tag and the amino acid substitution E954Q, which confers resistance to tetrodotoxin (TTX) and saxitoxin (STX), making it possible to block the endogenous CNS sodium channels and characterize the functional properties of just the transgenic Nav1.1 channels.

Section snippets

Ethical approval

All experiments were performed according to guidelines established by and with the approval of the Institutional Animal Care and Use Committees of Emory University and the University of California, Irvine.

Identification of a suitable BAC clone

Examination of the assembled mouse genomic sequence from the Mouse Genome Resources database (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) and the Ensembl Mouse genome server (http://www.ensembl.org/Mus_musculus/) revealed several overlapping BAC clones that contained sequences from the

Generation and characterization of control and mutant transgenic mice

Since we wanted a model system to study the mechanism by which SCN1A missense mutations lead to GEFS+, we constructed transgenic mice using a BAC clone containing the complete mouse Scn1a gene. One advantage of this approach is that the channel protein can be modified to distinguish it from endogenous channels, while the gene remains regulated by the native promoter in the BAC transgene. Because GEFS+ is inherited in an autosomal dominant fashion, even a single mutant allele in the presence of

Discussion

We have created a model for studying the pathophysiology of sodium channel dysfunction in GEFS+ by expressing the Nav1.1 R1648H mutation in transgenic mice. We observed no significant differences in the overall properties of sodium channels in transgenic and wild-type bipolar neurons in the absence of STX, however small differences in sodium channel gating were detected by isolating the mutant transgenic channels in the presence of STX. The larger population of endogenous wild-type Nav1.1, Nav

Acknowledgments

This study was supported by NIH Research Grants NS046484, NS051834 (A.E.) and NS48336 (A.L.G.), and grants from the March of Dimes Birth Defects Foundation (#5-FY02-250) (A.E.) and the McKnight Endowment Fund for Neuroscience (A.L.G.), the European Integrated Project EPICURE (M.M.) and the Italian Telethon grant GGP07277 (M.M.). K.D. was supported by a fellowship from the Epilepsy Foundation. L.P. was supported fellowships from AFIP and FAPESP (07-50534-5). ST was supported by FAPESP

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