Abstracts

Rationale: Familial epilepsies are often caused by mutations of voltage-gated Na+ channels, but correlation genotype-phenotype is not clear yet. In particular, the cause of the phenotypic variability observed in some epileptic families is unclear. We studied the Nav1.1 sodium channel α subunit M1841T mutation identified in a Generalized Epilepsy with Febrile Seizures plus (GEFS+) family characterized by a particularly large phenotypic spectrum, in order to disclose the origin of this variability.Methods: We transfected human tsA-201 cell line with human Nav1.1 cDNA and a reporter gene (Yellow Fluorescent Protein) to select transfected cells. When phenytoin was used, cells were incubated with the drug after the transfection for 36-48h. Interacting protein cDNA (sodium channel beta subunits, calmodulin or G-protein beta-gamma subunits) was cotransfected with Nav1.1 and reporter cDNAs and recordings were done after 36-48h. We recorded Na+ currents with the whole-cell configuration of the patch-clamp technique. The extracellular solution contained (mM): NaCl 140, CaCl2 2, MgCl2 2, HEPES 10, pH = 7.4 with NaOH. The pipette solution contained (mM): NMDG 195, NaCl 10, MgCl2 4, EGTA 5, Hepes 10, pH =7.2 with H3PO4. Results: We found that the Nav1.1 M1841T mutant is a loss of function because when expressed alone in human tsA-201 cells the current was no greater than background. Function was restored by incubation at temperature <30°C, as it is typical for trafficking defective mutant proteins. In most cases, trafficking defects are caused by the inability of the mutant protein to fold into a conformation that can pass the quality control system of the Endoplasmic Reticulum and by its subsequent degradation; incubation at low temperature probably rescues these mutants by decreasing the kinetics of folding. Trafficking defective mutants can also be rescued by interactions with other molecules (pharmacological chaperones). In fact, we found that incubation with 100uM phenytoin was able to partially rescue the mutant. Interestingly, out data show that also molecular interactions with modulatory proteins can restore function, because cotransfection with sodium channel beta subunits, calmodulin or G-protein beta-gamma subunits rescued the mutant.Conclusions: We show that a sodium channel trafficking defect can be the pathogenic mechanism of GEFS+ mutations and that interactions with accessory subunits or modulatory proteins can rescue the mutant channel. These interactions may modify the effect of the trafficking defective mutations in vivo and thus the phenotype. Variability in the strength of the interactions may be one of the causes of the phenotypic variability in the affected individuals of GEFS+ families and of the incomplete penetrance of GEFS+ mutations. Interacting drugs may be used to rescue the mutant in vivo.