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(v)2.1, 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(v)2.1 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(v)2.1 clustering at endoplasmic reticulum-plasma membrane junctions, K(v)2.1-G379R was expressed in HEK293T cells. K(v)2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of K(v)2.1-G379R with K(v)2.1-wild-type lowered induction of these structures relative to K(v)2.1-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(v)2.1 protein, as well as associated K(v)2.2 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.