Functional analysis of three Na_v1.6 mutations causing early infantile epileptic encephalopathy

The voltage-gated sodium channel Na_v1.6 is associated with more than 300 cases of epileptic encephalopathy. Na_v1.6 epilepsy-causing mutations are spread over the entire channel's structure and only 10% of mutations have been characterized at the molecular level, with most of them being gain of function mutations. In this study, we analyzed three previously uncharacterized Na_v1.6 epilepsy-causing mutations: G214D, N215D and V216D, located within a mutation hot-spot at the S3-S4 extracellular loop of Domain1. Voltage clamp experiments showed a 6-16 mV hyperpolarizing shift in the activation mid-point for all three mutants. V216D presented the largest shift along with decreased current amplitude, enhanced inactivation and a lack of persistent current. Recordings at hyperpolarized potentials indicated that all three mutants presented gating pore currents. Furthermore, trafficking experiments performed in cultured hippocampal neurons demonstrated that the mutants trafficked properly to the cell surface, with no significant differences regarding surface expression within the axon initial segment or soma compared to wild-type. These trafficking data suggest that the disease-causing consequences are due to only changes in the biophysical properties of the channel. Interestingly, the patient carrying the V216D mutation, which is the mutant with the greatest electrophysiological changes as compared to wild-type, exhibited the most severe phenotype. These results emphasize that these mutations will mandate unique treatment approaches, for normal sodium channel blockers may not work given that the studied mutations present gating pore currents. This study emphasizes the importance of molecular characterization of disease-causing mutations in order to improve the pharmacological treatment of patients.