The SCN8A p.(Gly1625Arg) variant associated with developmental and epileptic encephalopathy causes complex biophysical changes and reduced neuronal firing

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel Nav1.6, result in a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with Developmental epileptic encephalopathy (DEE), presenting with moderate epilepsy and severe developmental delay. This variant is positioned within the S4 segment of domain IV, a critical region for Nav1.6 function where pathogenic variants were shown to cause either a loss or gain of channel function, but often with mixed biophysical alterations. Voltage-clamp analyses of Nav1.6G1625R demonstrated a heterogeneous mixture of gain- and loss-of-function properties, including reduced current amplitudes, a marked increase in the time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Further studies using current clamp analyses in transfected cultured neurons showed that these intricate biophysical properties had a minor effect on neuronal excitability when firing relayed on both the endogenous and transfected Nav channels. Conversely, there was a marked reduction in the number of action potentials when the firing was driven solely by the transfected TTX-resistant mutant Nav1.6 compared to WT Nav1.6 channels. Moreover, using a computational model of mature cortical neurons, we revealed a mild reduction of neuronal firing when mimicking the patients heterozygous SCN8A expression. Structural modeling of Nav1.6G1625R, using the cryo-EM structure of Nav1.6, suggested the possible formation of cation-pi interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis demonstrated that this functional interaction affects the voltage dependence of inactivation in Nav1.6G1625R. Together, our analyses demonstrate a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain. ### Competing Interest Statement The authors have declared no competing interest.