Abstracts

Rationale: Mutations in the KCNT1 gene, encoding a sodium-activated potassium channel were identified in two patients suffering from different early-onset epilepsy syndromes [R474H: epilepsy of infancy with migrating focal seizures (EIMFS) and R474C: Ohtahara Syndrome (OS)]. EIMFS patients present with multiple seizure types occurring as early as 6 months age. OS, a rare epileptic syndrome renders many seizure types, with tonic seizures taking predominance, before age 3 months. Previous literature showed the inhibitory effects of quinidine on some mutant KCNT1 channels from patients. This study aims to determine the biophysical properties of two different variants affecting the same amino acid and leading to a distinctive clinical phenotype and assess the effect of a single dose of quinidine on these two KCNT1 mutations. Methods: Using the Xenopus laevis expression system, WT and mutant KCNT1 cRNA (R474H and R474C) was injected into Stage IV mature oocytes and incubated for 14-48 hours. Electrophysiological recordings were performed on an automated two-electrode voltage system (Roboocyte, Multi Channel Systems), before and after the application of 300 μM quinidine. Two-way ANOVA with Bonferroni correction was used to determine any significant differences in current-voltage relationships (I/V). Unpaired t-test was used to determine significance in percent of remaining current after treatment with quinidine Results: Both R474H and R474C generated larger current traces as compared to WT, indicating gain of function. Comparison of R474H [n=20] and WT [n=37] I/V curves showed a 1.5-fold increase in maximum current amplitude, with significant changes [**p≤0.01] in R474H channels between -40 mV and +80 mV. A 4-fold increase in maximum current amplitude was observed in R474C [n=20] channels, where significant changes [*p≤0.05] in the I/V curves compared to WT were observed from -60 mV. Application of quinidine reduced maximum current amplitudes in WT and mutant channels, however significant reduction in current (*p≤ 0.05) was only seen towards the positive potentials in WT and R474C I/V curves. The percentage of remaining current after application of quinidine uncovered significant variation in quinidine sensitivity with WT being most sensitive, followed by R474C and R474H, being the least sensitive to quinidine. Conclusions: The results indicate similar gain of function effect caused by different amino acid exchanges affecting the same position in the KCNT1 channel and leading to a different clinical phenotype and the variation in quinidine sensitivity for the two mutants. Funding: -