Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials in excitable tissues. These heteromultimeric complexes are comprised of a large (∼260 kDa) pore-forming α-subunit and smaller (∼30 kDa) β accessory subunits. The α-subunit is comprised of four homologous domains (DI-DIV) and exhibits significant homology to voltage-gated potassium and calcium channels. Recent structural studies have shown functional voltage-sensing and central pore subdomains within the predicted four-fold pseudo-symmetrical DI-DIV arrangement (Fig. 1A) (Catterall, 2000). Abnormal biophysical activity caused by mutations within channel subdomains is a common theme in inherited channelopathies, and epilepsy in particular.

Objective Pathogenic variants in KCNB1, encoding the voltage‐gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell‐surface expression. Methods We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high‐throughput functional assays. Specifically, we investigated the biophysical properties and cell‐surface expression of variant KV2.1 channels expressed in heterologous cells using high‐throughput automated electrophysiology and immunocytochemistry–flow cytometry. Results Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild‐type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell‐surface expression. Interpretation Our study establishes a platform for rapid screening of KV2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899–912

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 ...