Abstracts

Rationale: Epilepsy affects more than 3 million people in the United States, and more than two-thirds of cases have a presumed genetic origin. Despite the significant progress towards uncovering the pathophysiology of genetic epilepsy, a significant proportion of cases are treatment refractory, resulting in severe acute and chronic neurological consequences. Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel Kv2.1, were reported as a cause of early infantile epileptic encephalopathy type 26. Prior functional studies of KCNB1 variants indicated a range of mechanisms by which variants affect channel function. To date, reported effects include loss of voltage-sensitivity, loss of ion selectivity, and reduced surface expression. We have ascertained additional cases with KCNB1 epileptic encephalopathy variants to profile the spectrum of channel dysfunction.  Methods: Eleven KCNB1 missense mutations were introduced into a full-length human Kv2.1 cDNA expression construct by site-directed mutagenesis. We assessed the biophysical properties and protein trafficking of variant Kv2.1 channels in a heterologous expression system using automated electrophysiology and flow cytometry, respectively.  Results: Evaluation of variants as homotetramers or in a heterotetrameric configuration with wild-type Kv2.1 revealed diverse significant functional defects including altered current density, (n=21-55 per variant) and shifts in the voltage-dependence of activation and/or inactivation (n=14-30 per variant; p<0.05 vs. WT). Quantification of protein expression by flow cytometry also identified several variants with lower total cellular expression or impaired cell-surface trafficking.  Conclusions: Most KCNB1 variants (9/11) displayed mixed phenotypes with a general trend toward variable degrees of loss-of-function. The accelerated identification of epilepsy associated variants necessitates high-throughput evaluation of variants to assess the full spectrum of functional and biochemical defects. Ultimately, uncovering the full range of functional defects will aid in defining genotype-phenotype relationships, and could suggest targeted strategies for therapeutic intervention.  Funding: NINDS R01-NS053792