Functional Effects of Epilepsy Associated KCNT1 Mutations Suggest Pathogenesis via Aberrant Inhibitory Neuronal Activity

Abstract

KCNT1 (K+ channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the KCNT1 gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.

Modelling human KCNT1-epilepsy inDrosophila: a seizure phenotype and drug responses

“…While this preferential action on mutant KCNT1 channels is a highly desirable property for a KCNT1 therapeutic, it is unclear why this might be the case. It is possible that CBD binding to KCNT1 channel is state-dependent and the higher open probability of the mutant KCNT1 channels results in a higher efficacy of the drug 8 .…”

“…Almost all KCNT1 epilepsy mutations are heterozygous and missense, predicted to change a single amino acid in the protein 3 and de novo mutations most often account for severe cases. All mutations analysed in vitro to date (apart from T314A 8 ) significantly increase K + currents in comparison to normal KCNT1 channels [8][9][10][11][12] . There is evidence suggesting that increased K + currents in inhibitory neurons may be associated with (which was not certified by peer review) is the author/funder.…”

“…As in humans, the major inhibitory neurotransmitter in the Drosophila central nervous system is 3 -aminobutyric acid (GABA) 26 . Previous studies have suggested that reduced activity of inhibitory GABAergic neurons may be associated with seizures 8,13,14 . The mutant KCNT1 channels were expressed in different neural tissues, including GABAergic neurons, and investigated for a seizure phenotype using bang sensitive behavioural assays 27,28 .…”

“…The copyright holder for this preprint this version posted April 13, 2023. ; https://doi.org/10.1101/2023.04.11.536495 doi: bioRxiv preprint KCNT1-related seizures 8,13,14 . Reducing the activity of inhibitory neurons may explain how neuronal hyperexcitability, the mechanism underlying seizures, occurs in KCNT1-epilepsy.…”

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.…”