High throughput Characterization ofKCNB1variants Associated with Developmental and Epileptic Encephalopathy

Pathogenic variants inKCNB1, encoding the voltage-gated potassium channel Kv2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number ofKCNB1variants 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. We evaluated a series of 17KCNB1variants associated with DEE or neurodevelopmental disorder (NDD) 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. 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 co-expressed with wild-type Kv2.1. Quantification of protein expression also identified variants with reduced total Kv2.1 expression or deficient cell-surface expression.Our study establishes a platform for rapid screening of functional defects ofKCNB1variants associated with DEE and other NDDs, which will aid in establishingKCNB1variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.