Epilepsy and neurobehavioral abnormalities in mice with aKCNB1pathogenic variant that alters conducting and non-conducting functions of K_V2.1

AbstractDevelopmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay and are at a higher risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, includingKCNB1. The voltage-gated potassium channel KV2.1, encoded byKCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons and cardiomyocytes. Thede novopathogenic variantKCNB1-p.G379R was identified in an infant with epileptic spasms, atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected clustering at endoplasmic reticulum-plasma membrane junctions KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-WT lowered induction of these structures relative to KV2.1-WT alone, suggesting a dominant negative effect. To model this variantin vivo, we introducedKcnb1G379Rinto mice using CRISPR/Cas9 genome editing. We characterized neurological and neurobehavioral phenotypes ofKcnb1G379R/+(Kcnb1R/+) andKcnb1G379R/G379R(Kcnb1R/R) mice, and screened for cardiac abnormalities. Immunohistochemistry studies on brains fromKcnb1+/+(WT), Kcnb1R/+andKcnb1R/Rmice revealed genotype-dependent differences in the levels and subcellular localization of KV2.1, with reduced plasma membrane expression of the KV2.1-G379R protein, consistent within vitrodata.Kcnb1R/+andKcnb1R/Rmice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. In addition, bothKcnb1R/+andKcnb1R/Rmice exhibited abnormal interictal EEG abnormalities, including isolated spike and slow waves. Spontaneous seizure events were observed inKcnb1R/Rmice during exposure to novel environments and/or handling, while bothKcnb1R/+andKcnb1R/Rmutants were more susceptible to induced seizures.Kcnb1R/+andKcnb1R/Rmice exhibited prolonged rate-corrected QT interval on surface ECG recording. Overall, theKcnb1G379Rmice recapitulate many features observed in individuals with DEE due to pathogenic variants inKCNB1. This new mouse model ofKCNB1associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.