A novel strategy using cardiac sodium channel polymorphic fragments to rescue trafficking-deficient SCN5A mutations

Abstract

Background: Brugada syndrome (BrS) is associated with mutations in the cardiac sodium channel (Na(v)1.5). We previously reported that the function of a trafficking-deficient BrS Na(v)1.5 mutation, R282H, could be restored by coexpression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Na(v)1.5, spanning the H558R polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations.

Methods and results: Whole-cell patch clamping revealed that cotransfection in human embryonic kidney (HEK293) cells of the R282H channel with either the 40- or 20-amino acid cDNA fragments of Na(v)1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence resonance energy transfer suggested that the trafficking-deficient R282H channel was misfolded, and this was corrected on coexpression with R558-containing peptides that restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R polymorphism with 8 additional BrS Na(v)1.5 mutations with reduced currents and demonstrated that the peptide was able to restore significant sodium currents in 4 of them.

Conclusions: In the present study, we demonstrate that small peptides, spanning the H558R polymorphism, are sufficient to restore the trafficking defect of BrS-associated Na(v)1.5 mutations. Our findings suggest that it might be possible to use short cDNA constructs as a novel strategy tailored to specific disease-causing mutants of BrS.

Figure 1

Schematic Representation of the α-Subunit of hNav1.5. Ovals represent the locations of the mutations characterized in this study. The lower panel shows the sequence of the fragments that were created by amplifying a piece of either the wild-type (H558) or the polymorphic channel (R558).

Figure 2

Peptides Containing the H558R-Polymorphism Restore Function of the R282H BrS Mutant Channel A. Whole-cell sodium current traces recorded from transfected HEK293 cells. The R282H mutation expressed alone does not generate functional sodium current. Current was restored when the mutant was co-expressed with R558-containing peptide fused with CFP (R282H+R558-40aa and R282H+R558-20aa), but not when co-expressed with the WT peptide (R282H+H558-20aa). B. Peak sodium current densities. Current density was similar for WT (n=6), R282H+R558-40aa (n=8), and R282H+R558-20aa (n=12) and significantly lower for R282H+H558-20aa (n=11). C. Current-Voltage Relationships were similar for all conditions. *P<0.05 compared to WT.

Figure 3

Biophysical Properties of Rescued Currents are Altered A. Conductance was similar for WT, rescued channels (R282H+R558-40aa and R282H+R558-20aa), and WT channels treated with the R558-containing peptide (WT+R558-40aa). B. Steady-State Inactivation of rescued currents (closed symbols) was significantly shifted toward depolarized potentials. C. Recovery from Inactivation. The time constants of recovery from inactivation were significantly faster for rescued channels. D. Time Constant of Inactivation. Inactivation of R282H+R558-20aa channels (n=9) was significantly slower than WT channels (n=7). R282H+R558-40aa channels (n=5) and WT+R558-40aa channels (n=9) behaved similarly to WT. *P<0.05 compared to WT.

Figure 4

Biochemical Analysis A. Representative Western blot of three independent experiments showing WT and mutant proteins in total cell lysates in the presence or absence of the R558-containing peptide (R558-20aa). NT, non-transfected cells. B. Representative surface biotinylation experiments to investigate the absence or presence of the R282H mutant in the cell membrane with and without the peptide containing the polymorphism. Three independent biotinylation experiments were performed. C. Quantification normalized to WT. Data are presented as mean±SEM. *P<0.05 compared to WT.

Figure 5

FRET in Normal and Mutant Channels A. Diagram of FRET construct containing CFP fused to the C-terminus of hNa_v1.5 and YFP inserted into the Domain II-III linker at amino acid position 1022. B. Currents elicited from the FRET construct are similar to WT currents. C. FRETc values for YFP-1022 (n=43) were similar to YFP-1022+R558-40aa (n=35), YFP-1022+R558-20aa (n=39), YFP-1022/R282H+R558-40aa (n=43), YFP-1022/R282H+R558-20aa (n=46), and YFP-1022/R282H+R558-20aa+Brefeldin A (n=34). FRETc of YFP-1022/R282H (n= 45) alone, YFP-1022/R282H+H558-40aa (n=36), and YFP-1022/R282H+H558-20aa (n=42) were significantly smaller than WT. *P<0.05 compared to WT.