Functional evaluation of epilepsy‐associated KCNT1 variants in multiple cellular systems reveals a predominant gain of function impact on channel properties

Abstract

ObjectiveGain of function variants in the sodium‐activated potassium channel KCNT1 have been associated with pediatric epilepsy disorders. Here, we systematically examine a spectrum of KCNT1 variants and establish their impact on channel function in multiple cellular systems.MethodsKCNT1 variants identified from published reports and genetic screening of pediatric epilepsy patients were expressed in Xenopus oocytes and HEK cell lines. Variant impact on current magnitude, current–voltage relationships, and sodium ion modulation were examined.ResultsWe determined basic properties of KCNT1 in Xenopus oocyte and HEK systems, including the role of extra‐ and intracellular sodium in regulating KCNT1 activity. The most common six KCNT1 variants demonstrated strong gain of function (GOF) effects on one or more channel properties. Analysis of 36 total variants identified phenotypic heterogeneity but a strong tendency for pathogenic variants to exert GOF effects on channel properties. By controlling intracellular sodium, we demonstrate that multiple pathogenic KCNT1 variants modulate channel voltage dependence by altering the sensitivity to sodium ions.SignificanceThis study represents the largest systematic functional examination of KCNT1 variants to date. We both confirm previously reported GOF channel phenotypes and expand the number of variants with in vitro GOF effects. Our data provide further evidence that novel KCNT1 variants identified in epilepsy patients lead to disease through generalizable GOF mechanisms including increases in current magnitude and/or current–voltage relationships.

KCNT1 Channel Blockers: A Medicinal Chemistry Perspective

“…At the molecular level, different mechanisms underlying the enhanced channel functioning of GoF KCNT-1 variants have been proposed, among which are the following: the suppression of subconductance states together with changes in protein kinase C regulation [ 49 ]; changes in Na + sensitivity [ 55 ]; increased channel cooperative gating together with decreased single-channel conductance [ 56 ]; and altered interactions with binding partners [ 57 , 58 ]. Considering the GoF variants as the most pathogenic KCNT1 variants, during recent years, several efforts have been made by both academia and industry to discover KCNT1 blockers suitable as therapeutic tools for precision medicine.…”

In Silico Assisted Identification, Synthesis, and In Vitro Pharmacological Characterization of Potent and Selective Blockers of the Epilepsy-Associated KCNT1 Channel

“…21−23 More than 60 distinct pathogenic variants have been reported in KCNT1, the vast majority of which prompt gainof-function (GoF) effects on KCNT1 channels, resulting in enhanced potassium currents when studied in heterologous expression systems in vitro. 24,25 Different molecular mechanisms leading to enhanced channel function have been identified, including: (1) changes in Na + sensitivity; 25 (2) increased channel cooperative gating; 26 (3) suppression of subconductance states; 14 (4) changes in protein kinase C regulation; 14 and (5) altered interactions with binding partners. 27,28 Seizures in patients with KCNT1-related epilepsy, particularly those with EIMFS or DEE phenotypes, are often highly refractory to pharmacological therapy.…”

Heterozygous expression of a Kcnt1 gain-of-function variant has differential effects on SST- and PV-expressing cortical GABAergic neurons

“…Consistent with this role, loss-of-function (LOF) studies using mouse models lacking KCNT1, and the associated sodium-activated potassium (K Na ) current, have shown enhanced AP firing across multiple neuron types (Evely et al, 2017 ;Liu et al, 2022 ;Lu et al, 2015 ;Martinez-Espinosa et al, 2015 ;Reijntjes et al, 2019 ;Zhang et al, 2022 ). Characterizations of pathogenic DEEassociated KCNT1 variants in heterologous cells found that nearly all cause gain-of-function (GOF) effects on the channel, increasing the associated K Na current (Hinckley et al, 2023 ;Kim et al, 2014 ;McTague et al, 2018 ;Milligan et al, 2014 ;Tang et al, 2016 ). Based on LOF studies, this would be expected to reduce neuronal excitability; however, it is difficult to predict the effects of these GOF variants on AP generation in neurons, particularly among neuronal subtypes, a priori.…”