EPILESSIA E CANALI VOLTAGGIO DIPENDENTI DEL SODIO: MECCANISMI PATOGENETICI.

The SCN1A gene, coding for the isoform Nav1.1 of voltage gated sodium channels, is the main target of epileptogenic mutations. Mutations in this gene have been identified in about 80% of patients affected by Severe Myoclonic Epilepsy of Infancy (SMEI), in about 10% of patients suffering from Generalized Epilepsy with Febrile Seizures Plus (GEFS+). Nav1.1 mutations that cause GEFS+ are missense mutations, whereas those that give rise to the much more severe disorder SMEI can either be missense ones or result in truncated channels that are predicted to be non-functional. Several evidences point to loss of function as the main effect of Nav1.1 mutations. It is puzzling that loss of function mutations in Nav1.1 lead to epilepsy, a disorder characterized by brain hyperexcitability; however, data from Nav1.1 knock out and knock in mice indicate that Nav1.1 is the predominant isoform in at least some types of inhibitory interneurons. Previous data on mutant of Cav1.2, Nav1.2 and Nav1.5 suggest that truncated proteins can exert dominant-negative effects, contributing to more severe phenotypes. Therefore we hypothesized that also SMEI truncation mutants could have dominant-negative effects, explaining the more severe phenotype in comparison with GEFS+. In the first project we have studied the effects of the SMEI mutations R222X and R1234X on hNav1.1, hNav1.2 and hNav1.6. Patch-clamp analysis of coexpressed mutants and wild type channels in tsA201 cells showed that both truncated mutant proteins had no effect on the expression of wild type channels; however, the truncated proteins modified the gating properties of Nav1.1 and Nav1.6, worsening their loss of function. It is known that some Nav1.1 missense mutation cause loss of function because of folding defects. We have previously demonstrated that two mutations located in the C-terminus cause loss of function inducing folding defects, thus retention in the endoplasmic reticulum and probably degradation (Rusconi et al., 2007 and 2009). Moreover, we have shown that the folding of the mutant channel can be stabilized by the co-expression of interacting proteins or incubation with pharmacological chaperones, leading to a partial restoration of the expression on the plasma membrane and therefore to a partial recovery of its activity. It is possible to speculate that differences in the expression levels or the presence of polymorphisms in the accessory proteins could influence the interaction with the misfolded channel, inducing a differential degree of rescue which possibly determines the severity of the pathology and therefore the phenotypic variability. In the second project, to understand if misfolding could be considered a common pathogenic mechanism for this type of epilepsy, we screened and functionally tested other epileptogenic Nav1.1 mutants, found in families with high phenotypic variability. We performed the functional characterization with whole cell voltage clamp recordings of tsA-201 cells transfected with the mutant channel. To date, we have found three potential new folding defective mutants. We obtained partial or almost complete rescue of these mutants by incubation at lower temperature and by incubation with pharmacological chaperones or with interacting proteins. The identified mutations are localized in different domains of the channel. Thus, mutations causing folding defects are not restricted to the C-terminal intracellular domain and may be common in Nav1.1 related epilepsies.