Elsevier

Heart Rhythm

Volume 13, Issue 5, May 2016, Pages 1113-1120
Heart Rhythm

SCN5A(K817E), a novel Brugada syndrome–associated mutation that alters the activation gating of NaV1.5 channel

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Background

Brugada syndrome (BrS) is an inherited lethal arrhythmic disorder characterized by syncope and sudden cardiac death from ventricular tachyarrhythmias. Here we identified a novel K817E mutation of SCN5A gene in a man with type 1 BrS electrocardiogram pattern using next-generation sequencing targeted for 73 cardiac disorder-related genes. SCN5A encodes the α-subunit of NaV1.5 voltage-gated Na+ channel, and some of its mutations are linked to BrS. The proband had no mutation in any of the other arrhythmia-related genes sequenced.

Objective

We investigated whether the K817E mutation causes a functional change of NaV1.5 channel responsible for the BrS phenotype.

Methods

We compared the electrophysiological properties of the whole-cell currents mediated by wild-type and mutant channels heterologously expressed in human embryonic kidney 293 cells by using a voltage-clamp technique.

Results

The K817E mutation reduced the Na+ current density by 39.0%–91.4% at membrane potentials from −55 to −5 mV. This reduction resulted from a ~24-mV positive shift in the voltage dependence of activation. The mutation also decelerated recovery from both fast and intermediate inactivation, whereas it had little effect on the cell surface expression, single-channel conductance, voltage-dependence of fast inactivation, entry into intermediate inactivation, use-dependent loss of channel availability, or closed-state inactivation.

Conclusion

The K817E mutation of SCN5A gene leads to loss of function of NaV1.5 channel and may underlie the BrS phenotype of the proband.

Introduction

The SCN5A gene encodes the pore-forming α-subunit of the human cardiac voltage-gated Na+ channel NaV1.5, which mediates the fast inward Na+ current (INa) that contributes to the rapid depolarization of the cardiac action potential.1, 2 Mutations in SCN5A gene may cause cardiac Na+ channelopathies, sometimes leading to sudden cardiac death. Such channelopathies include Brugada syndrome (BrS), long QT syndrome type 3, and dilated cardiomyopathy.3, 4, 5, 6 BrS is an inherited cardiac arrhythmia disorder possibly underlying the development of ventricular fibrillation and leading to sudden cardiac death in patients with structurally normal hearts.7 Patients with BrS have a characteristic electrocardiogram (ECG) pattern with ST-segment elevation at the right precordial leads.8, 9 Some SCN5A mutations previously identified in patients with BrS cause the loss of function of NaV1.5 channel by reducing the surface expression level of the channel or changing the voltage and time dependences of channel activation and/or inactivation.4, 5, 10, 11, 12

Here we report a functional study of a NaV1.5 channel involving a novel SCN5A(K817E) mutant that was identified in a 38-year-old man. NaV1.5 channel is composed of 4 domains, each of which has 6 membrane-spanning hydrophobic helical segments. K817 is located in the fourth segment of domain 2 (D2S4). D2S4 serves as a voltage sensor and its positively charged amino acids including K817 are considered important for voltage sensing of the channel.13 The BrS-related mutations identified in the positively charged amino acids in the D2S4 region reportedly affect the voltage dependence of fast inactivation.14, 15 Similarly, a substitution of K817 with the negatively charged glutamate may affect channel gating. To assess this possibility, we performed functional analyses of the mutant NaV1.5 channel in a heterologous expression system of human embryonic kidney 293 cells.

Section snippets

Plasmid construction

To express the α-subunit of NaV1.5 channel, wild-type (WT) human SCN5A complementary DNA (GenBank: NM_198056) was subcloned into pReceiver-M12 vector containing the N-terminal 3× FLAG epitope (GeneCopoeia, Rockville, MD) [FLAG-SCN5A(WT)]. FLAG-SCN5A(K817E) was generated on the basis of FLAG-SCN5A(WT) using a site-directed mutagenesis kit (Toyobo, Osaka, Japan). To express the β-subunit of NaV1.5 channel, WT SCN1B complementary DNA (GenBank: NM_001037) was subcloned into pReceiver-M12 vector

Clinical and genetic background

In a school physical examination, a 10-year-old girl (Figure 1A, IV-1) reported that she has a couple of relatives who suffered from a cardiac defect or died of sudden cardiac death. Based on this report, the hospital asked her family members, including the proband (her father, Figure 1A, III-1), for further investigation. The proband’s ECG showed a type 1 BrS pattern consisting of a spontaneous coved-type ST-segment elevation and T-wave inversion in leads V1-V2 (Figure 1B). Although the

Discussion

We have identified a novel K817E mutation of SCN5A in a man with type 1 BrS ECG pattern. The proband had no mutation in any of the other major arrhythmia-related ion channels. As to the genes not responsible for arrhythmia, there was a missense mutation in VCL gene. VCL encodes vinculin, a cytoskeletal protein associated with cadherin-mediated cell-cell junctions and integrin-mediated cell-matrix adhesions.24 Defects in VCL may cause cardiomyopathies by modifying the interaction between

Conclusion

In an asymptomatic man with type 1 BrS ECG pattern, we identified a novel SCN5A mutation causing a K817E substitution in D2S4 voltage sensor of NaV1.5 channel. Functional analyses revealed that the mutation reduced the INa density by a marked positive shift in the voltage dependence of activation and by slowing down recovery from both fast and intermediate inactivation. In contrast, the mutation had little effect on the cell surface expression level, single-channel conductance, the voltage

Acknowledgments

We thank the provider of the gene sample for his kind cooperation.

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  • Cited by (0)

    Dr Kinoshita and Mr Takahashi contributed equally to this work.

    This study was partly supported by KAKENHI from Japan Society for the Promotion of Science (grant no. 24790745, to Dr Kinoshita), (grant nos. 24590852 and 15k08867, to Dr Hata; grant no. 26430012, to Dr Tabata), Japan Agency for Medical Research and Development (AMED) (grant no. 15dk020710h0002, to Dr Tabata), and Presidential Discretionary Funds, University of Toyama 2014 (to Dr Nishida).

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