On the Feasibility of Quantifying Sodium Channel Na v 1.6 Protein in Mouse Brain using targeted UHPLC-ESI- MRM Mass Spectrometry.

RATIONAL: Nav 1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Nav 1.6 expression in animal models. This research explores the feasibility of quantifying Nav 1.6 expression in mouse brains using targeted multiple reaction monitoring mass spectrometry.

METHODS: A combination of in-silico tryptic Nav 1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work-up including plasma membrane isolation, trypsin-based proteolysis and UHPLC-ESI-MS/MS to detect the presence of Nav 1.6 in induced HEK293 cells. The unique Nav 1.6 peptide, DSLFIPR, was selected as probe for quantifying Nav 1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8aN1768D/+ and heterozygous null Scn8a+/- mice using isotope dilution targeted mass spectrometry.

RESULTS: The feasibility of using targeted MRM for quantifying Nav 1.6 expression in mice brains was demonstrated. Expression of Nav 1.6 in brains (hippocampi) from wild type and mutant Scn8aN1768D/+ mice were found to be around 0.40 fmoles/μg. Mutant null Scn8a+/- heterozygous mice showed, on the other hand, showed levels of 0.22 fmoles/μg as expected based on this particular mutation which only generates fifty-percent of the expression in wild type mice. Nav 1.6-overexpressed HEK293 cells showed 3.7 fmoles/μg of Nav 1.6 expression, suitable of screening new compounds for Nav 1.6 blocking activity.