Abstract
B1-C121W is down but Not Out: Epilepsy-Associated Scn1b-C121W Results in a Deleterious Gain-of-Function
Kruger LC, O'Malley HA, Hull JM, Kleeman A, Patin GA, Isom LL. J Neurosci 2016;36:6213–6224.
Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β subunits play nonconducting roles as cell adhesion molecules, which allow them to function in cell–cell communication, neuronal migration, neurite outgrowth, neuronal pathfinding, and axonal fasciculation. Mutations in SCN1B, encoding VGSC β1 and β1B, are associated with epilepsy. Autosomal-dominant SCN1B-C121W, the first epilepsy-associated VGSC mutation identified, results in genetic epilepsy with febrile seizures plus (GEFS+). This mutation has been shown to disrupt both the sodium-current-modulatory and cell-adhesive functions of β1 subunits expressed in heterologous systems. The goal of this study was to compare mice heterozygous for Scn1b-C121W (Scn1b+/W) with mice heterozygous for the Scn1b-null allele (Scn1b+/−) to determine whether the C121W mutation results in loss-of-function in vivo. We found that Scn1b+/W mice were more susceptible than Scn1b+/− and Scn1b+/+ mice to hyperthermia-induced convulsions, a model of pediatric febrile seizures. β1-C121W subunits are expressed at the neuronal cell surface in vivo. However, despite this, β1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. β1-C121W subcellular localization is restricted to neuronal cell bodies and is not detected at axon initial segments in the cortex or cerebellum or at optic nerve nodes of Ranvier of Scn1bW/W mice. These data, together with our previous results showing that β1-C121W cannot participate in trans-homophilic cell adhesion, lead to the hypothesis that SCN1B-C121W confers a deleterious gain-of-function in human GEFS+ patients.
Commentary
Brain voltage-gated sodium channels exist as a heterotrimeric complex consisting of a large pore-forming a subunit and two different β subunits (β1/β3 and β2/β4). The β subunits are single pass transmembrane proteins that contain an extracellular Ig loop that is homologous to motifs present in the Ig superfamily of cell adhesion molecules. β1 and β3 interact noncovalently with the α subunit, while β2 and β4 interact with the α subunit via a covalent interaction. The β subunits perform multiple functions as part of the voltage-gated sodium channel complex, including promoting plasma membrane trafficking, modulating biophysical properties, and acting as cell-adhesion molecules (1).
Many mutations in voltage-gated sodium channel complex genes have been associated with various types of epilepsy encompassing a wide range of clinical severity. The first voltage-gated sodium channel mutation, reported in 1998, was found in SCN1B, encoding the voltage-gated sodium channel β1 subunit. The autosomal dominant mutation SCN1B-C121W was identified in a large family with generalized epilepsy with febrile seizures plus (GEFS+) type 1 (2). The SCN1B-C121W mutation affects a highly conserved cysteine residue that normally forms a disulfide bridge that is critical for maintaining the Ig loop. Subsequently, other heterozygous mutations in SCN1B have been identified, with the majority located in the Ig loop domain (1).
Although it was first reported almost 20 years ago, there is still active investigation into the mechanisms of how the SCN1B-C121W mutation leads to epilepsy. The initial report demonstrated that the coexpression of the mutant β1-C121W and α subunits in Xenopus oocytes resulted in biophysical differences in sodium conductance that suggested loss of its modulatory effect on the α subunit (2). Similar effects were observed in mammalian cell lines (3–5). Additionally, the SCN1B-C121W mutation results in the loss of cell-adhesion properties, while plasma membrane trafficking functions are preserved (3–7). Studies of the mutation effect in heterologous systems have been informative; however, they do not reveal the complete picture of how the SCN1B-C121W mutation acts within the nervous system.
More recent studies have addressed this issue using knock-in mouse models. Neurons are highly polarized cells with many important functional subdomains. A domain of particular importance for neuronal excitability and epilepsy is the axon initial segment (AIS), which is the site of action potential initiation. AIS function is highly dependent on high density clustering of many ion channels, including voltage-gated sodium channels. Recent studies demonstrated that the Scn1b-C121W mutation disrupts AIS localization of β1, with reduced AIS expression in heterozygous Scn1b+/W mice and absence in Scn1bW/W mice (8, 9). In addition, Scn1b+/W mice have increased susceptibility to hyperthermia-induced seizures, as well as temperature-sensitive increases in excitability measured by slice electrophysiology (8, 10). Although several groups postulated that the Scn1b-C121W mutation results in loss of β1 function, it was unclear if this was the whole story.
In the current study, Kruger and colleagues directly compared the Scn1b+/W mice with heterozygous null Scn1b+/− mice to determine whether Scn1b-C121W is consistent with a loss-of-function allele. First, they examined hyperthermia-induced seizure susceptibility and observed that young Scn1b+/W mice are more susceptible than Scn1b+/− mice, with a reduced latency, lower threshold temperature, and elevated severity. The experiments were carefully conducted, with all mice on the same strain background and wild-type littermate controls from each line, and support a modest phenotypic difference between Scn1b+/W and Scn1b+/− mice. This observation suggested that the C121W mutation may not be a simple loss-of-function.
Follow-up biochemical experiments showed that β1-C121W was expressed at lower levels and was not fully glycosylated compared with wild-type. This finding was demonstrated in brain membranes prepared from Scn1b+/W and Scn1b+/+ mice. The amount of β1 protein in the heterozygous Scn1b+/W samples was approximately half of that observed in wild-type controls, and migrated at a lower molecular weight. PNGaseF digestion to remove N-linked glycosylation ameliorated the apparent molecular weight difference, indicating incomplete glycosylation of the β1-C121W mutant protein. The reduction in β1 protein remained consistent across a range of ages from postnatal day 14 through ≥ 20 weeks of age. Using cell surface biotinylation assays, they demonstrated that the mutant β1-C121W protein was present at the cell surface. However, immunocytochemistry experiments showed that β1-C121W was excluded from specialized sites of enriched wild-type β1 expression, including the AIS and nodes of Ranvier. Exclusion of β1-C121W from the AIS was observed in multiple brain regions, including cortical layers II and IV and cerebellar Purkinje neurons. Furthermore, coimmunoprecipitation experiments demonstrated that the mutant β1-C121W did not associate with sodium channel α subunits.
Based on their findings, the authors propose that the Scn1b+/W mutation is not a simple loss-of-function but rather a gain-of-function. They speculate that presence of the mutant β1-C121W protein on the soma may effectively dilute the density of wild-type β1 subunits and reduce their function. Based on this postulation, it may be more accurate to designate Scn1b+/W as a dominant negative or antimorphic mutation. Regardless of semantics, this study reiterates the essential role of the β1 subunit in neuronal function and suggests that the amount of functional β1 may be a critical determinant of neuronal excitability. This suggests the possibility that natural variation in the level of β1 expression may buffer some individuals against deleterious effects of heterozygous SCN1B mutations, and may explain some of the variable penetrance and expressivity observed in families with SCN1B mutations. Additionally, it suggests that upregulation of the wild-type allele may have therapeutic benefit for SCN1B epilepsy mutations.