Seizure Phenotype and Underlying Cellular Defects in Drosophila Knock-In Models of DS (R1648C) and GEFS+ (R1648H) SCN1A Epilepsy.

Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A. R1648C is associated with the severe disorder Dravet Syndrome, and R1648H, with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R-R control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced lifespans and spontaneous and temperature-induced seizures not observed in R-R controls. Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared to R-R controls. Since this change is constitutive it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity associated with these mutations in humans could be due in large part to differences in genetic background. SIGNIFICANCE STATEMENT Knock-in Drosophila lines were generated by CRISPR-Cas9 gene editing to explore how the R1648C and R1648H mutations in the SCN1A sodium channel gene contribute to distinct epilepsy disorders, Dravet syndrome and GEFS+ respectively. Drosophila heterozygous for the R-C or R-H mutation displayed spontaneous seizures exacerbated at high temperature. GABAergic neurons in both mutants exhibited sustained depolarizations, interrupting neuronal firing, that increased in incidence at elevated temperature. A hyperpolarized deactivation threshold in R-C and R-H sodium currents was constitutive implicating interaction with other heat-sensitive processes to alter firing properties. The similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines suggests that disorders of different severity in humans could be due in large part to differences in genetic background.