Common human SCN5A polymorphisms have altered electrophysiology when expressed in Q1077 splice variants

Background

Eight common (>0.5%) polymorphisms of SCN5A have been described in the US population. Every human also continuously generates two wild-type (WT) splice variants, one with a glutamine residue at position 1077 (Q1077) and one lacking this glutamine (Q1077del). One polymorphism (H558R) has been studied in both splice variants, five polymorphisms (R34C, R481W, S524Y, P1090L,V1951L) have not been previously studied, and two polymorphisms (S1103Y and R1193Q) have been studied in only one of the two splice variants.

Objectives

The purpose of this study was to examine the electrophysiologic molecular phenotype of the eight common polymorphisms in the two human splice variants of SCN5A.

Methods

Currents from 16 channels (all polymorphisms in both splice variants) were determined by voltage clamp and compared with WT after expression in HEK-293 cells.

Results

Six of eight polymorphisms showed a distinct phenotype that depended upon the background splice variant used for expression. Only R34C and V1951L showed no functional differences. S524Y showed a dramatic reduction in current density in the Q1077 background similar to that previously described for H558R. Four other polymorphisms (R481W, P1090L, S1103Y, R1193Q) showed shifts in activation, inactivation, or recovery that depended upon the splice variants. Shifts of a similar magnitude have been reported for arrhythmia syndrome mutations and are thought to be pathogenic.

Conclusion

The majority of common human SCN5A polymorphisms have a distinct molecular phenotype that depends upon the splice variant background. These findings have implications for the interpretation of previous studies of arrhythmia mutations. The significance of these findings for clinical arrhythmia remains to be elucidated.

Introduction

The human cardiac Na channel gene SCN5A encodes the ion channel hNa_v1.5,¹ which carries inward Na current (I_Na) in the heart. This channel is responsible for a large peak I_Na that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and specialized conduction tissue (Purkinje cells and others) and for a late I_Na that influences repolarization and refractoriness. Mutations in SCN5A that cause various heritable arrhythmia syndromes, including congenital long QT syndrome subtype 3 (LQT3) and type I Brugada syndrome,2, 3 are relatively rare. Mutations above a certain threshold allelic frequency (e.g., >0.5%) in healthy subjects in a population are called common polymorphisms. Polymorphisms in genes other than SCN5A have been implicated in phenotypic variability in physiology, pharmacology, and pathophysiology.4, 5 The prevalence and ethnic specificity of eight common polymorphisms of SCN5A have been described (Figure 1).⁶ The polymorphisms (with allelic frequencies) are R34C (9.2% blacks, 1.7% Hispanic), R481W (0.6% blacks, 0.5% Hispanic), S524Y (3.3% blacks), H558R (29% blacks, 20.4% whites, 9.2% Asians, 23.1% Hispanics), P1090L (2.2% Asians), S1103Y (6.5% blacks), R1193Q (0.3% whites, 8.0% Asians), and V1951L (6.7% Hispanic). Of these polymorphisms, only three (H558R,7, 8, 9 S1103Y,¹⁰ R1193Q¹¹) have been characterized by electrophysiologic study. Interestingly, all three polymorphisms studied had functional effects: S1103Y was linked to increased arrhythmia risk in the black population,¹⁰ R1193Q was linked to LQT3,¹¹ and H558R modulated the effects of other arrhythmia mutations.8, 9

We previously described two splice variants of SCN5A⁷ in human heart resulting in expression of two distinct hNa_v1.5 sodium channels: one comprising 2,016 amino acids and containing a glutamine at position 1077 (Q1077) and one having only 2,015 amino acids because of the absence of this amino acid (Q1077del). Transcripts for these two splice variants were present in every human heart examined in an approximate transcript ratio of 2:1. Approximately two thirds of the transcript encoded the major 2,015-amino-acid variant lacking Q1077 (Q1077del) and one third encoded the minor 2,016-amino-acid variant with Q1077. Therefore, any allele with a mutation at any site would be expressed in the context of both splice variants. We previously showed that H558R expressed in the two splice variant backgrounds had profoundly different function, with H558R in the Q1077 background showing an expression defect or “loss of function.” This is the only SCN5A mutation to date that has been studied in both backgrounds. In this study, we examined all eight polymorphisms in both backgrounds and compared them with the two wild-type (WT) variants in order to answer two questions. (1) Do the other five common polymorphisms also have distinct functional phenotypes compared with WT channels? (2) Does the splice variant background affect the functional phenotype ascribed to a polymorphism? The answers to these questions have implications for the interpretation of previous studies of arrhythmia mutations in heterologous systems and may have implications for human arrhythmia susceptibility.