A common SCN5A polymorphism modulates the biophysical defects of SCN5A mutations

Abstract

Background: Defects in the cardiac sodium channel gene, SCN5A, can cause a broad spectrum of inherited arrhythmia syndromes. After genotyping of a proband who presented with syncope, the SCN5A mutant P2006A and the common polymorphism H558R were identified.

Objective: The main objective of this study was to determine whether the SCN5A-H558R polymorphism could modify the defective gating kinetics observed in the P2006A mutation and therefore explain why this gain-of-function mutation has been identified in control populations.

Methods: Mutations were engineered using site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. Whole-cell sodium currents were measured at room temperature using the whole-cell patch-clamp technique.

Results: In HEK293 cells, P2006A displayed biophysical defects typically associated with long QT syndrome by increasing persistent sodium current, producing a depolarizing shift in voltage dependence of inactivation, and hastening recovery from inactivation. Interestingly, when coexpressed either on the same or different genes, P2006A and H558R displayed currents that behaved like wild type (WT). We also investigated whether H558R can modulate the gating defects of other SCN5A mutations. The H558R polymorphism also restored the gating defects of the SCN5A mutation V1951L to the WT level.

Conclusions: Our results suggest that H558R might play an important role in stabilization of channel fast inactivation and may provide a plausible explanation as to why the P2006A gain-of-function mutation has been identified in control populations. Our results also suggest that the SCN5A polymorphism H558R might be a disease-modifying gene.

Figure 1

Pedigree of a family with a SCN5A mutation, location and topology of SCN5A mutations. A, Pedigree shows affected individuals. While the mother (I-1) was homozygous for H558R, the father (I-2) was heterozygous for H558R and P2006A. The proband (II-2) and her brother (II-1) were homozygous for H558R and heterozygous for P2006A. All of them carry the H558R polymorphism on the same allele as P2006A. B, Sequence analysis of SCN5A reveals a change of Histidine to Arginine at position 558 and a change of Proline to Alanine at position 2006. C, Topological diagram of sodium channel showing amino acid residues where polymorphism and mutations occur respectively.

Figure 2

Representative whole-cell sodium current traces from representative experiments of SCN5A-WT, SCN5A-P2006A, SCN5A-H558R-P2006A, SCN5A-H558R + SCN5A-P2006A, and SCN5A-H558R + SCN5A-H558R-P2006A.

Figure 3

Representative traces of late I_Na for SCN5A-WT (black, panel A and B), SCN5A-P2006A (light gray, panel A), SCN5A-H558R-P2006A (gray, panel A), SCN5A-H558R+SCN5A-P2006A (gray, panel B), and SCN5A-H558R+SCN5A-H558R-P2006A (light gray, panel B). A, TTX-sensitive persistent sodium currents for SCN5A-WT, SCN5A-P2006A mutation, and SCN5A-P2006A mutation with the sodium channel polymorphism H558R. As expected, the P2006A mutation displays incomplete inactivation. However, in the presence of the H558R polymorphism, the P2006A mutation’s inactivation kinetics are restored to the WT level. B, Superimposition of the normalized sodium currents recorded from SCN5A-WT, SCN5A-H558R+ SCN5A-P2006A, and SCN5A-H558R+SCN5A-H558R-P2006A elicited by clamping at −30 mV. Neither SCN5A-H558R+SCN5A-P2006A nor SCN5A-H558R+SCN5A-H558R-P2006A mutants alter the persistent current compared to WT channels. The right traces represent the magnified regions outlined by the boxes to highlight the persistent sodium currents of the left panels. The dotted lines indicate zero current. Statistically significant differences in late I_Na comparing mutant channels with SCN5A-WT are indicated (*P<0.05, student t-test).

Figure 4

Electrophysiological characterization of SCN5A mutations. A, Voltage dependence of activation for SCN5A-P2006A (n=12), SCN5A-H558R-P2006A (n=20), SCN5A-H558R+SCN5A-P2006A (n=6), and SCN5A-H558R+SCN5A-H558R-P2006A (n=6) in which currents were compared to SCN5A-WT (n=12). B, Steady-state inactivation recorded using the standard protocol as shown in inset and fitted to a Boltzmann distribution. The midpoints of inactivation (V_1/2) in mutant channels of SCN5A-P2006A were significantly shifted toward depolarized voltages compared to WT channels. C, Mutant channels of SCN5A-P2006A display faster recovery from inactivation than WT channels.

Figure 5

Electrophysiological characterization of SCN5A mutations. A, Voltage dependence of activation for SCN5A-V1951L (n=6), SCN5A-H558R+SCN5A-V1951L (n=6) in which currents were compared to SCN5A-WT (n=6). B, The midpoints of steady-state inactivation (V_1/2) in mutant channels of SCN5A-V1951L were significantly shifted toward depolarizing voltages compared to WT channels. C, Neither SCN5A-V1951L nor SCN5A-H558R+SCN5A-V1951L mutants modify the time course of recovery from inactivation. D, Superimposition of the normalized sodium currents recorded from SCN5A-WT (black), SCN5A-V1951L (gray), and SCN5A-H558R + SCN5A-V1951L (light gray). The transient sodium currents obtained during depolarization to −30 mV from a holding potential of −120 mV were normalized to illustrate similarity in fast inactivation. Neither SCN5A-V1951L nor SCN5A-H558R + SCN5A-V1951L mutants alter the late sodium current compared to WT channels. The dotted lines indicate zero current.