Variants in the KCNT1 gene, which encodes a Na+-activated K+ channel, cause several severe childhood epilepsy disorders that are largely refractory to treatment. Previously, pathogenic KCNT1 variants were shown to increase channel current when assayed in non-neuronal cells, leading to the fundamental question of how increasing a current that is usually associated with dampening neuronal excitability leads to disorders characterized by excessive, synchronous neuronal activity. We have created two mouse models with orthologous pathogenic human KCNT1 variants that have frequent seizures. In both models, cortical inhibitory neurons (cINs) show strong impairments in membrane excitability and action potential generation, whereas excitatory neurons (cENs) do not. These data suggest that KCNT1 gain-of-function (GOF) variants cause childhood epilepsy by impairing the function of cINs, and that KCNT1 channels play cell-type-specific roles in regulating neuronal excitability. This proposal will test these hypotheses by (1) determining how KCNT1 block and GOF affect membrane excitability of cIN subtypes, (2) using newly-developed KCNT1-selective inhibitors to measure the current and how it is altered by disease-causing variants in cIN subtypes, and (3) testing how impaired membrane excitability in subpopulations of cINs affects their activity in vivo, and how this relates to seizures or cortical hyperexcitability. Determining these vulnerable cell types, the underlying mechanisms, and the in vivo effects of their dysfunction will allow us to relate cellular deficits to epileptiform activity and seizures. The results will advance our understanding of the regulation of cIN excitability, the physiological roles of the KCNT1-mediated current, and the disease mechanisms of KCNT1 GOF in an in vivo model of epilepsy. This will narrow the knowledge gap between the biophysical effects of ion channel variants and the resulting dysfunction of networks, and has the potential to improve the targeting of precision therapies for severe childhood epilepsies.