Functional and Biochemical Analysis of a Sodium Channel β1 Subunit Mutation Responsible for Generalized Epilepsy with Febrile Seizures Plus Type 1

Abstract

Generalized epilepsy with febrile seizures plus type 1 is an inherited human epileptic syndrome, associated with a cysteine-to-tryptophan (C121W) mutation in the extracellular immunoglobin domain of the auxiliary beta1 subunit of the voltage-gated sodium channel. The mutation disrupts beta1 function, but how this leads to epilepsy is not understood. In this study, we make several observations that may be relevant for understanding why this beta1 mutation results in seizures. First, using electrophysiological recordings from mammalian cell lines, coexpressing sodium channel alpha subunits and either wild-type beta1 or C121Wbeta1, we show that loss of beta1 functional modulation, caused by the C121W mutation, leads to increased sodium channel availability at hyperpolarized membrane potentials and reduced sodium channel rundown during high-frequency channel activity, compared with channels coexpressed with wild-type beta1. In contrast, neither wild-type beta1 nor C121Wbeta1 significantly affected sodium current time course or the voltage dependence of channel activation. We also show, using a Drosophila S2 cell adhesion assay, that the C121W mutation disrupts beta1-beta1 homophilic cell adhesion, suggesting that the mutation may alter the ability of beta1 to mediate protein-protein interactions critical for sodium channel localization. Finally, we demonstrate that neither functional modulation nor cell adhesion mediated by wild-type beta1 is occluded by coexpression of C121Wbeta1, arguing against the idea that the mutant beta1 acts as a dominant-negative subunit. Together, these data suggest that C121Wbeta1 causes subtle effects on channel function and subcellular distribution that bias neurons toward hyperexcitabity and epileptogenesis.

Functional reciprocity between Na + channel Na v 1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

“…Scn1b (β1) null mice are ataxic, experience spontaneous seizures, and exhibit a prolonged cardiac QT interval, demonstrating that β1 modulates electrical excitability in vivo (12,13). Consistent with this, human mutations in SCN1B result in epilepsy and arrhythmia (14)(15)(16)(17)(18)(19)(20)(21). As a member of the Ig superfamily of cell adhesion molecules (CAMs), β1 mediates cellular aggregation, cytoskeletal recruitment, and extracellular matrix interactions in vitro (11).…”

“…Given that Scn1b mutations result in channelopathies in vivo (12)(13)(14)(15)(16)(17)(18)(19)(20)(21), and that β1-mediated neurite outgrowth in CGNs requires I Na and Na v 1.6, we postulated that β1 might regulate electrical excitability in the cerebellum. To test this, we recorded APs in P12-13 WT and Scn1b null CGNs in cerebellar slices by whole-cell patch clamping.…”

Sodium Channel β1 Subunits Promote Neurite Outgrowth in Cerebellar Granule Neurons

“…Although the observed seizure activity may be caused by altered sodium channel isoform expression in the hippocampus, our present results suggest that errors in neurite extension and pathfinding may also contribute. A human mutation in SCN1B that causes generalized epilepsy with febrile seizures plus type 1 results in the replacement of a key cysteine residue in the ␤1 Ig loop domain with tryptophan (36,37). We have shown that, in addition to inhibiting ␤1-mediated channel modulation, this mutation abolishes ␤1-mediated homophilic cell adhesive interactions (37).…”

Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Nav1.1 Na+Channel Mutant

“…Lymphocyte T-killer CD8 receptor, which we used as negative control, has a similar structure (Cole and Gao, 2004). The GEFSϩ C121W mutation of ␤1, which we used in some of the experiments, destroys the intramolecular disulfide bridge and inhibits ␤1 function (Wallace et al, 1998;Meadows et al, 2002) and is depicted as a hollow circle. ␤2 and ␤4 subunits are covalently linked to the ␣ subunit by disulfide bridges (dashed-dotted line), but the cysteine residues implicated have not been identified yet.…”

“…1 and Fig. 5C) destroys a disulfide bridge in the extracellular Ig-loop, and the mutant is a loss of function, the capacity of which to modulate ␣ subunit functions and to establish extracellular protein-protein interactions is impaired (Wallace et al, 1998;Meadows et al, 2002). Coexpression of hNa v 1.1-M1841T and ␤1C121W mutants induced a 2.6-fold increase in current density (Fig.…”