Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Abstract

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

Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations

“…A recent paper on Kv2.1-related neurodevelopmental disorders documented a spectrum of Kv2.1 mutations (50). Among pathogenic mutations that did not affect current density, negative shifts in the voltage dependence of inactivation in the order of 10 to 20 mV were observed that would sufficiently reduce channel availability, resulting in a loss of function (50).…”

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

“…HEK293T cells were transfected with WT rat K v 2.1 in pRBG4, rat K v 2.1 S586A in pCGN, HA-tagged WT human K v 2.1, or HA-tagged WT human K v 2.1 with the G379R mutation ( Shi et al, 1994 ; Lim et al, 2000 ; Kang et al, 2019 ). Cells were transiently transfected using Lipofectamine 2000 following the manufacturer’s protocol within 18 h of plating.…”

“…In 30–50% of infants diagnosed with an epileptic encephalopathy, a causative mutation has been identified in a known epilepsy gene ( McTague et al, 2016 ). Previous work has identified heterozygous de novo KCNB1 pathogenic variants in individuals with DEE ( Torkamani et al, 2014 ; Saitsu et al, 2015 ; Thiffault et al, 2015 ; Allen et al, 2016 ; Calhoun et al, 2017 ; de Kovel et al, 2017 ; Latypova et al, 2017 ; Marini et al, 2017 ; Miao et al, 2017 ; Bar, 2020 ; Kang et al, 2019 ). In addition to seizures, individuals with KCNB1 variants display comorbidities that include developmental delay, intellectual disability, features of autism spectrum disorder, ADHD and aggression, as well as borderline long QT syndrome in some cases ( Calhoun et al, 2017 ; de Kovel et al, 2017 ; Marini et al, 2017 ; Bar, 2020 ).…”

“…Several KCNB1 variants have been functionally characterized in vitro and shown to exhibit reduced K v 2.1 conductance, as well as altered ion selectivity, expression and localization ( Torkamani et al, 2014 ; Saitsu et al, 2015 ; Thiffault et al, 2015 ; Kang et al, 2019 ). K V 2.1-G379R channels exhibited loss of voltage-sensitivity and ion-selectivity, resulting in a non-specific cation leak current ( Torkamani et al, 2014 ).…”

Association of small-fiber polyneuropathy with three previously unassociated rare missense SCN9A variants

“…The function of the affected protein is also important, and neuronal proteins that influence action potentials such as SCN9A have such critical roles that even minor alterations can be deleterious or fatal. 43 Some genetic risk modifiers may only become relevant in individuals also exposed to other environmental or genetic risks or in specific ethnicities or locations. 44 – 46 Recent mutations (sporadic cases with no family history) and those with incomplete penetrance are also underestimated.…”