Epilepsy and neurobehavioral abnormalities in mice with a dominant-negative KCNB1 pathogenic variant

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay, and often have elevated risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel K_V2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons. In addition to its canonical role as a voltage-gated potassium conductance, K_V2.1 also serves a highly conserved structural function organizing endoplasmic reticulum-plasma membrane junctions clustered in the soma and proximal dendrites of neurons. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, and 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 K_V2.1 clustering at endoplasmic reticulum-plasma membrane junctions, K_V2.1-G379R was expressed in HEK293T cells. K_V2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of K_V2.1-G379R with K_V2.1-wild-type lowered induction of these structures relative to K_V2.1-WT alone, consistent with a dominant negative effect. To model this variant in vivo, we introduced Kcnb1^G379R into mice using CRISPR/Cas9 genome editing. We characterized neuronal expression, neurological and neurobehavioral phenotypes of Kcnb1^G379R/+ (Kcnb1^R/+) and Kcnb1^G379R/G379R (Kcnb1^R/R) mice. Immunohistochemistry studies on brains from Kcnb1^+/+, Kcnb1^R/+ and Kcnb1^R/R mice revealed genotype-dependent differences in the expression levels of K_V2.1 protein, as well as associated K_V2.2 and AMIGO-1 proteins. Kcnb1^R/+ and Kcnb1^R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. Spontaneous seizures were observed in Kcnb1^R/R mice, as well as seizures induced by exposure to novel environments and/or handling. Both Kcnb1^R/+ and Kcnb1^R/R mutants were more susceptible to proconvulsant-induced seizures. In addition, both Kcnb1^R/+ and Kcnb1^R/R mice exhibited abnormal interictal EEG activity, including isolated spike and slow waves. Overall, the Kcnb1^G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1-associated 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.