Epilepsy, characterized by recurrent spontaneous seizures, affects over 50 million people worldwide and is one of the most common neurological disorders. Mutations in the voltage-gated sodium channel gene, SCN8A, have recently been identified as an important cause of severe pediatric epilepsy. With the advent of next-generation sequencing, over 150 pathogenic SCN8A mutations have been identified in patients. At least twenty of these mutations are located in the S4 voltage-sensor domains (VSDs) that are critical for voltage-dependent gating of the SCN8A channel protein, Nav1.6. Patients with VSD mutations exhibit a broad spectrum of clinical phenotypes, including intellectual disability, ataxic gait, hypotonia, and treatment-resistant epilepsy. Some patients also present with mild or no seizures but with neurodevelopmental abnormalities such as autism, intellectual disability and developmental delay. To better understand how mutations in the Scn8a VSDs can cause this broad range of clinical phenotypes, we utilized CRISPR/Cas9 to knock-in human SCN8A epilepsy mutations into the mouse Scn8a DIIS4 and DIVS4 VSDs. One unplanned but exciting consequence of this strategy was the generation of four mouse lines with small in-frame insertions/deletions in Scn8a: [ΔRVF] a 9bp deletion in DIIS4 that removes three amino acid residues (R848_F850del), [849D] a 3bp insertion in DIIS4 that introduces one amino acid residue (R848_V849_InsD), [ΔVIR] a 9bp deletion in DIVS4 that removes three amino acid residues (V1616_R1618del), and [ΔIRL] a 9bp deletion in DIVS4 that removes three amino acid residues (I1617_L1619del). Since most published SCN8A mutations are missense mutations, these mouse lines will provide a unique opportunity to 1) contribute to our understanding of the range of clinically-relevant phenotypes associated with SCN8A dysfunction, 2) further our understanding of the impact of mutations in the VSD on SCN8A function, and 3) identify phenotypes associated with small in-frame SCN8A insertions/deletions. We hypothesize that this class of SCN8A mutations (small insertions and deletions) will lead to a range of seizure and behavioral phenotypes. It is also possible that distinct phenotypes might be associated with these types of mutations when compared to point mutations. Consistent with this, our preliminary data demonstrate that heterozygous ΔRVF mutants exhibit increased resistance to induced seizures, whereas heterozygous ΔVIR mutants exhibit increased seizure susceptibility and spontaneous seizures. In addition, homozygous ΔRVF mutants exhibit reduced nerve conduction velocity, while nerve conduction velocity was unaltered in homozygous Scn8a null mice. Thus, the goal of this R03 proposal is to characterize and compare the seizure and behavioral phenotypes of these four Scn8a mouse lines. In Aim 1, we will establish the effect of each mutation on seizure susceptibility. In Aim 2, we will determine the effect of each mutation on mouse behavior and mot...