Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Na_v1.1 Na⁺Channel Mutant

Abstract

Familial epilepsies are often caused by mutations of voltage-gated Na ϩ channels, but correlation genotype-phenotype is not yet clear. In particular, the cause of phenotypic variability observed in some epileptic families is unclear. We studied Na v 1.1 (SCN1A) Na ϩ channel ␣ subunit M1841T mutation, identified in a family characterized by a particularly large phenotypic spectrum. The mutant is a loss of function because when expressed alone, the current was no greater than background. Function was restored by incubation at temperature Ͻ30°C, showing that the mutant is trafficking defective, thus far the first case among neuronal Na ϩ channels. Importantly, also molecular interactions with modulatory proteins or drugs were able to rescue the mutant. Protein-protein interactions may modulate the effect of the mutation in vivo and thus phenotype; variability in their strength may be one of the causes of phenotypic variability in familial epilepsy. Interacting drugs may be used to rescue the mutant in vivo.

A rescuable folding defective Nav1.1 (SCN1A) sodium channel mutant causes GEFS+: Common mechanism in Nav1.1 related epilepsies?

“…We introduced the mutation c.5746A>G (R1916G; R1927G in the longer hNa v 1.1 splice variant) by means of the Quick Change XL kit (Stratagene) using pCDM8-hNa v 1.1 as template and the primers 5'-GTGCTTACGGGCGCCACCTTTTAAAGC (forward) and 5'-AAAGGTGGCGCCCGTAAGCACGC (reverse). The cDNAs of human voltage gated Na + channel β1 and β2 subunits were provided by Dr. Al George (Vanderbild University, Nashville, TN); we subcloned them into the bicistronic plasmids pIRES-YFP and pIRES-CFP (Clontech) in order to express with the same plasmid both the protein of interest and the fluorescent protein as reporter (Rusconi et al, 2007). We cloned human β3 and β4 subunits from tsA-201 cells and subcloned them into pIRES-YFP as described in (Rusconi et al, 2007).…”

“…The cDNAs of human voltage gated Na + channel β1 and β2 subunits were provided by Dr. Al George (Vanderbild University, Nashville, TN); we subcloned them into the bicistronic plasmids pIRES-YFP and pIRES-CFP (Clontech) in order to express with the same plasmid both the protein of interest and the fluorescent protein as reporter (Rusconi et al, 2007). We cloned human β3 and β4 subunits from tsA-201 cells and subcloned them into pIRES-YFP as described in (Rusconi et al, 2007). The cDNAs of G protein β2 and γ3 subunits were provided by Dr. Melvin Simon (Caltech, Pasadena, CA); we subcloned them into pIRES-YFP as described previously (Mantegazza et al, 2005b).…”

“…The cDNAs of G protein β2 and γ3 subunits were provided by Dr. Melvin Simon (Caltech, Pasadena, CA); we subcloned them into pIRES-YFP as described previously (Mantegazza et al, 2005b). The cDNA of rat calmodulin was provided by Dr. John Adelman (Vollum Institute, Portland, OR); we subcloned it into the bicistronic vector pIRES-dsRED2 (Clontech) as described previously (Rusconi et al, 2007). The plasmids expressing the two isoforms of FGF14 (pQBI-FGF14-1a and pIRES-GFP-FGF14-1b) were provided by Drs.…”

“…We have recently characterized a loss of function Na v 1.1 folding defective mutant (M1841T) found in a GEFS+ family with extreme phenotypic variability that ranged from mild FS to SMEI (Rusconi et al, 2007). Foldingdefective mutants are recognized as incorrectly folded proteins by the quality control system of the endoplasmic reticulum and degraded (Bernier et al, 2004).…”

“…mild febrile seizures) whereas inefficient interactions should worsen the loss of function generating severe phenotypes (e.g. SMEI) (Rusconi et al, 2007;Stafstrom, 2008).…”