A Novel Epilepsy Mutation in the Sodium ChannelSCN1AIdentifies a Cytoplasmic Domain for β Subunit Interaction

Abstract

A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFSϩ). The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the C-terminal cytoplasmic domain of the sodium channel ␣ subunit. The mutation decreased modulation of the ␣ subunit by ␤1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes. There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of ␤1. This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp. Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus. These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity. Direct interaction between the cytoplasmic C-terminal domain of the wild-type ␣ subunit with the ␤1 or ␤3 subunit was first demonstrated by yeast two-hybrid analysis. The SCN1A peptide K1846-R1886 is sufficient for ␤ subunit interaction. Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the ␣ and ␤1 subunits. The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility.

Pathogenic mechanism of recurrent mutations of SCN8A in epileptic encephalopathy

“…In addition to its role in channel inactivation, the C‐terminal domain of Na v 1.6 interacts with several proteins that regulate channel trafficking or activity 20, 23, 30. To determine whether mutations at Arg1872 disrupt these interactions, we tested the effect of the p.Arg1872Trp mutation with substitution of the large uncharged tryptophan residue.…”

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

“…␤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 ␤3 subunits have noncovalent intracellular and extracellular interactions with the ␣ subunit (dashed lines) (McCormick et al, 1998;Qu et al, 1999;Meadows et al, 2001;Spampanato et al, 2004). M1841T lies in a 41-residue area, highlighted in the inset, comprising a cytoplasmic binding domain for ␤1 and, probably, ␤3 (Spampanato et al, 2004).…”

“…␤1 and ␤3 subunits have noncovalent intracellular and extracellular interactions with the ␣ subunit (dashed lines) (McCormick et al, 1998;Qu et al, 1999;Meadows et al, 2001;Spampanato et al, 2004). M1841T lies in a 41-residue area, highlighted in the inset, comprising a cytoplasmic binding domain for ␤1 and, probably, ␤3 (Spampanato et al, 2004). The GEFSϩ mutation D1855Y (Spampanato et al, 2004) is also shown in the inset; we used a different numeration than the original D1866Y, because the clone that we used codifies for a shorter hNa v 1.1 variant (see Results).…”

“…M1841T lies in a 41-residue area, highlighted in the inset, comprising a cytoplasmic binding domain for ␤1 and, probably, ␤3 (Spampanato et al, 2004). The GEFSϩ mutation D1855Y (Spampanato et al, 2004) is also shown in the inset; we used a different numeration than the original D1866Y, because the clone that we used codifies for a shorter hNa v 1.1 variant (see Results). Part of the receptor site of intracellular pore blocker drugs is located in the S6 segment of domain IV (Catterall, 2000), ϳ70 aa upstream of M1841T; interaction with the antiepileptic drug phenytoin is depicted.…”

“…We studied the M1841T GEFSϩ mutation of Na v 1.1 ␣ subunit (hNa v 1.1-M1841T), a missense mutation that replaces with a threonine the methionine at position 1841, located in a region of the C-terminal cytoplasmic domain involved in interactions with accessory subunits (Spampanato et al, 2004) (see Fig. 1).…”