Abstracts

Rationale: Pathogenic variants in KCNT1, a gene encoding the sodium-gated potassium channel KNa1.1, account for ~40% of epilepsy of infancy with migrating focal seizures (EIMFS). Electrophysiological studies in heterologous expression systems support a channel gain-of-function in nearly all pathogenic variants, including KCNT1-L437F that we recently identified. We hypothesized that a gain-of-function KNa1.1 conductance with relatively greater dampening of excitability in inhibitory interneurons may underlie the pathogenicity of these variants. To assess the impact of the pathogenic channel on intrinsic neuronal properties, we generated human excitatory neurons from human induced pluripotent stem cells (hiPSCs) carrying the KCNT1-L437F variant for electrophysiological analysis in parallel with neurons from healthy sex-matched controls. Methods: Peripheral blood mononuclear cells (PBMCs) obtained from a patient with KCNT1-associated epilepsy heterozygous for the KCNT1-L437F variant were reprogrammed in parallel with control healthy sex-matched samples. To assess the electrophysiological impact of the KCNT1-L437F variant, hiPSC-derived neurons were induced into excitatory neurons using lentiviral overexpression of NGN2 prior to patch-clamp recording in current clamp mode at days in vitro (DIV) DIV21 and DIV35.  Results: Both passive membrane and active (action potential; AP) firing properties 'matured' between DIV21 and DIV35 timepoints to more closely approximate neurons from acute brain slices. At DIV21, KCNT1-L437F neurons exhibited a more hyperpolarized AP threshold and increased AP amplitude compared to control neurons. By DIV35, AP properties were no longer significantly different, but KCNT1-L437F neurons had a significantly lower input resistance, suggestive of larger membrane surface area. With depolarizing current injections, there was no significant difference in rheobase current needed to evoke an action potential, but KCNT1-L437F neurons 'failed' to fire at higher frequencies, as apparent in an inverted U-shaped AP frequency-intensity (F-I) curve. Conclusions: Human iPSC-derived excitatory neurons derived from a patient with KCNT1-associated epilepsy (p.L437F) are more likely to fire action potentials at DIV21, evidenced by a hyperpolarized AP threshold and increased AP amplitude. At DIV35, these neurons have a lower input resistance, characteristic of a homeostatic increase in membrane surface area. Understanding this developmental influence opens the door to targeting key mediators of developing cortical circuitry in patients with KCNT1-associated epilepsy. Funding: NINDS K08 (NS104237), NCATS KL2 (TR001424)