Abstract 498: SiRNA Silencing and Deletion of the Putative NAD Binding Site of the Novel Brugada Syndrome gene GPD1-L Increase Cardiac Sodium Current
Introduction: Mutations in the cardiac Na+ channel SCN5A cause ~20% of cases of Brugada syndrome, a rare inherited cause of arrhythmias and sudden death. We identified a mutation (A280V) in the Glycerol-3 Phosphate Dehydrogenase 1-Like gene (GPD1-L) in a large Brugada syndrome family. In HEK-293 cells that express wild type (WT) GPD1-L, transfection with A280V GPD1-L decreases SCN5A membrane expression and Na+ current (INa). GPD1-L contains a consensus NAD binding site (GxGxxG) and is 81% percent similar to GPD, an NAD binding gene with oxidoreductase activity. We therefore hypothesized that expression of WT GPD1-L alters cardiac Na+ currents and that the NAD binding site is crucial for its activity.
Methods: SiRNA specific for GPD1-L and negative control siRNA (Neg siRNA) constructs were designed. PCR based site-directed mutagenesis was used to create a deletion mutant of GPD1-L (Δ7–18) lacking the NAD binding site. GPD1-L siRNA and Neg siRNA were transfected in HEK cells stably expressing SCN5A and Δ7–18 plasmid was co-transfected with RFP. INa was recorded at 25°C using whole-cell voltage clamp.
Results: GPD1-L siRNA decreased GPD1-L mRNA by 86% (n = 9) vs. 9% for the Neg siRNA (n = 8, p < 0.02). Suppressing GPD1-L increased INa by 43% vs. control (at −20mV, from −329±33 pA/pF to −569±75 pA/pF, p = 0.01, Fig. 1A⇓). Deletion of the NAD binding site increased INa by 52 % (at −20 mV, from −206±29 pA/pF to −429±59 pA/pF, p<0.01, Fig. 1B⇓).
Conclusion: WT GPD1-L is a negative regulator of cardiac Na+ current, presumably by modulating membrane trafficking. The putative NAD binding site is required for this effect, suggesting an important role for oxidoreductase activity in Na+ channel trafficking.