Mutations in voltage-gated ion channels are one of the leading causes of monogenic epilepsies. A common feature of monogenic epilepsies is variable expressivity among family members with the same mutation, suggesting that genetic modifiers may influence susceptibility. The Scn2aQ54 transgenic mouse model of epilepsy has an epilepsy phenotype with strain-dependent expressivity. Kcnv2, which encodes the voltage- gated potassium channel subunit Kv8.2, was identified as a genetic modifier of the Scn2aQ54 phenotype. Screening of epilepsy patients identified two non-synonymous coding sequence variants that altered channel function in a heterologous expression system, suggesting that KCNV2 also contributes to human epilepsy susceptibility. Mouse data indicates that Kcnv2 is a quantitative modifier, with increased steady-state levels of Kcnv2 expression associated with an increase in Scn2aQ54 phenotype severity. These data suggest that regulatory sequence variation in the promoter and 3'UTR of KCNV2 may influence seizure susceptibility by altering steady-state expression levels. The proposed studies will characterize the Kcnv2 promoter and 3' UTR in mouse and human and determine the effects of mouse and human regulatory variants on Kcnv2 transcription rates and mRNA stability. In Specific Aim 1, rapid amplification of cDNA ends (RACE) analysis and RNase protection assays will be used to determine the transcription start site (TSS) region and 3'UTR organization of mouse and human KCNV2. Luciferase promoter assays will then be used to identify the minimal promoter regions. In Specific Aim 2, luciferase reporter constructs will be used to determine the effects of promoter sequence variation on mouse and human KCNV2 promoter activity. Tet-off reporter constructs will be used to test the influence of mouse and human 3' UTR sequence variation on mRNA stability. As a complementary approach, primary hippocampal cultures will be used to test the stability of mouse Kcnv2 mRNA following transcriptional inhibition. Next, the in vivo effects of selected Kcnv2 regulatory element(s) will be evaluated by BAC transgenesis. Elucidating the effects of regulatory sequence variants on Kcnv2 expression will help us to determine the molecular basis of the Kcnv2 modifier effect and advance our knowledge of the mechanisms by which genetic modifiers influence epilepsy susceptibility.