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