Abstracts

Rationale: Mutations in brain voltage-gated sodium channel genes have been associated with monogenic epilepsies having varying severity. Mutations in SCN2A, encoding Nav1.2, are associated with a range of disorders including the relatively mild disorder benign familial neonatal-infantile seizure (BFNIS), and more severe syndromes such as epilepsy of infancy with migrating focal seizures and early infantile epileptic encephalopathy type 11 (Ohtahara syndrome). Ohtahara syndrome is one of the earliest presenting age-dependent epileptic encephalopathies, with onset as early as the first ten days of life, and has a very poor prognosis with frequent death in early infancy. Ohtahara syndrome often presents with intractable seizures, and severe cognitive deficits. The early onset of some Nav1.2 associated epileptic encephalopathies suggest that developmentally regulated alternative splicing of Nav1.2, may be important to consider when determining the functional consequences of these variants. We hypothesized that the Nav1.2 mutations associated with Ohtahara syndrome may exhibit greater dysfunction when expressed in a splice variant expressed prominently during early development. Methods: We engineered four Ohtahara syndrome associated SCN2A variants (T236S, E999K, S1336Y, and T1623N), into the adult and neonatal isoforms of Nav1.2 and performed whole-cell patch clamp recording of tsA201 cells expressing either WT or mutant Nav1.2 along with the beta1 and beta2 accessory subunits. We examined whole-cell current density, voltage-dependence of activation and inactivation, recovery from inactivation, and persistent sodium current. Results: While most mutations showed current density values similar to wild-type channels, S1336Y showed a drastic reduction of current density in both the adult and neonatal isoforms. Interestingly, T236S showed a large increase in current density, but only in the neonatal isoform. Additionally, while T236S, E999K, and S1336Y, when engineered into the adult isoform, showed no difference in voltage dependence of activation compared to WT Nav1.2, when these mutations were engineered into the neonatal isoform they exhibited a large hyperpolarized shift in the activation curve compared to neonatal WT Nav1.2. These data indicated that alternative splicing impacts the functional consequences of some SCN2A variants. Finally, S1336Y and T1623N showed a depolarized shift in steady-state inactivation in both the adult and neonatal isoforms. None of the variants affected recovery from inactivation or persistent sodium current. Conclusions: Ohtahara syndrome associated Nav1.2 variants exhibit predominantly gain-of-function effects, including hyperpolarized shifts in voltage dependence of activation and depolarized shifts in voltage dependence of inactivation. Importantly, some variants showed more severe dysfunction in the neonatal splice isoform, suggesting that alternative splicing may act as an intrinsic, intragenic modifier of some early onset epilepsy syndromes. Funding: NIH NS032387