Korean J Physiol Pharmacol.  2024 Jul;28(4):313-322. 10.4196/kjpp.2024.28.4.313.

Requirement of βsubunit for the reduced voltage-gated Na⁺ current of a Brugada syndrome patient having novel double missense mutation (p.A385T/R504T) of SCN5A

• Park NK
• Choi SW
• Park SJ
• Woo J
• Kim HJ
• Kim WK
• Moon SH
• Park HJ
• Kim SJ

• KMID: 2557202
• DOI: http://doi.org/10.4196/kjpp.2024.28.4.313

Abstract

Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na⁺ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (I_Na) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of I_Na was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of I_Na and slower recovery from inactivation, consistent with the loss-of-function in Na⁺ channels. The SCN1B dependent reduction of I_Na was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.

Keyword

Cardiac arrhythmia; Brugada syndrome; Missense mutation; NAV1.5 voltage-gated sodium channel; Sodium channel beta subunit

Figure

• 

  Fig. 1 Twelve-lead electrocardiogram (ECG) of a patient with overlap syndrome with a double missense mutation in SCN5A. (A) Representative ECG showed a coved type ST-segment elevation in leads V2 (2 mm) and T-wave abnormality. (B) Representative ECG showed intraventricular conduction delay with prolonged QRS widening to 121 ms (normal value < 100 ms).

• 

  Fig. 2 p.A385T/R504T does not affect Na+ current (INa) density. (A) Schematic drawing of the cardiac voltage-gated Na+ channel α-subunit (NaV1.5) showing position of the A385T/R504T in the loop connecting transmembrane segments 5 and 6 in domain 1 (S5-S6 in DI) and segments 6 and 1 between domain 1 and 2 (DI-DII linker). (B) Representative whole-cell Na+ current recordings of WT and p.A385T/R504T. (C) Average Na+ current-voltage (I-V) relation for WT and p.A385T/R504T channels. (D) Voltage-dependence of activation for WT and p.A385T/R504T channels by normalizing peak conductance against the membrane voltage. WT, wild type.

• 

  Fig. 3 Characterization of Na+ channel property in the steady-state activation and inactivation. (A) Representative traces of voltage dependence of inactivation. The protocol is shown in left panel. (B) Voltage dependence of activation and inactivation and window current. The window current refers to the expanded segment of the overlapping region between the activation and inactivation of both the WT and p.A385T/R504T channels. (C) Time constant of recovery from inactivation to A385T/R504T was elicited with a double pulse protocol. (D) The relationship between P2/P1 and the inter-pulse interval was graphed, with curves representing double-exponential functions. The fast and slow time constants were estimated from the plotted double-exponential functions. WT, wild type; N.S, not significant.

• 

  Fig. 4 Reduced p.A385T/R504T INa co-transfected with β1-subunit. (A) Representative whole-cell Na+ current traces of WT and p.A385T/R504T in the presence of β1. (B) I-V relation of WT + β and p.A485T/R504T + β. (C) Comparison of peak INa density (p = 0.0027). p-value less than 0.05 was considered to be statistically significant, *p < 0.05; **p < 0.01. (D) Voltage dependence of activation for WT and p.A385T/R504T in the presence of β1-subunit measured form 12-20 cells. (E) Representative Western blotting image of NaV1.5 in transfected HEK293 cells with and without β1-subunit. WT, wild type; I-V, current-voltage; DB, double mutant; GFP, green fluorescent protein.

• 

  Fig. 5 Characterization of Na+ channel property in the steady-state activation and inactivation in co-expressed β1-subunit. (A) Representative traces of voltage dependence of inactivation in the presence of β1-subunit. (B) Voltage dependence of activation and inactivation, and window current. (C) Representative traces of time course of recovery from inactivation of WT and p.A385T/R504T with β1-subunit. (D) The P2/P1 ratio was plotted against the inter-pulse interval, and the resulting curves were fitted with double-exponential functions. The time constants for the fast (p = 0.0250) and slow components were estimated from these plotted functions. p-value less than 0.05 was considered to be statistically significant, *p < 0.05. WT, wild type; N.S, not significant.

• 

  Fig. 6 Whole-cell current recordings of p.A385T and p.R504T. Na+ channels were expressed by transfection in HEK293 cells in the presence of β1-subunit. (A) I-V relation of WT + β, p.A385T + β, and p.R504T + β. (B) Comparison of peak INa density. Compared to the WT group, p.R504T + β significantly reduced current density (p = 0.0167). *p < 0.05; **p < 0.01; ***p < 0.001. (C) Voltage dependence of activation and inactivation, and window current. (D) Time course of recovery from inactivation of WT, p.A385T, and p.R504T with β1-subunit. WT, wild type; N.S, not significant.

Reference

1. Napolitano C, Bloise R, Monteforte N, Priori SG. 2012; Sudden cardiac death and genetic ion channelopathies: long QT, Brugada, short QT, catecholaminergic polymorphic ventricular tachycardia, and idiopathic ventricular fibrillation. Circulation. 125:2027–2034. DOI: 10.1161/CIRCULATIONAHA.111.055947. PMID: 22529064.

2. Rook MB, Evers MM, Vos MA, Bierhuizen MF. 2012; Biology of cardiac sodium channel Nav1.5 expression. Cardiovasc Res. 93:12–23. DOI: 10.1093/cvr/cvr252. PMID: 21937582.

3. Wilde AAM, Amin AS. 2018; Clinical spectrum of SCN5A mutations: long QT syndrome, Brugada syndrome, and cardiomyopathy. JACC Clin Electrophysiol. 4:569–579. DOI: 10.1016/j.jacep.2018.03.006. PMID: 29798782.

4. Carmeliet E. 2002; Congenital LQT syndrome from gene to torsade de pointes. Korean J Physiol Pharmacol. 6:1–8.

5. Grant AO, Carboni MP, Neplioueva V, Starmer CF, Memmi M, Napolitano C, Priori S. 2002; Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation. J Clin Invest. 110:1201–1209. DOI: 10.1172/JCI0215570. PMID: 12393856. PMCID: PMC150793.

6. Remme CA, Wilde AA. 2008; SCN5A overlap syndromes: no end to disease complexity? Europace. 10:1253–1255. DOI: 10.1093/europace/eun267. PMID: 18820249.

7. Porretta AP, Probst V, Bhuiyan ZA, Davoine E, Delinière A, Pascale P, Schlaepfer J, Superti-Furga A, Pruvot E. 2022; SCN5A overlap syndromes: an open-minded approach. Heart Rhythm. 19:1363–1368. DOI: 10.1016/j.hrthm.2022.03.1223. PMID: 35351625.

8. Bezzina C, Veldkamp MW, van Den Berg MP, Postma AV, Rook MB, Viersma JW, van Langen IM, Tan-Sindhunata G, Bink-Boelkens MT, van Der Hout AH, Mannens MM, Wilde AA. 1999; A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res. 85:1206–1213. DOI: 10.1161/01.RES.85.12.1206. PMID: 10590249.

9. Remme CA, Wilde AA, Bezzina CR. 2008; Cardiac sodium channel overlap syndromes: different faces of SCN5A mutations. Trends Cardiovasc Med. 18:78–87. DOI: 10.1016/j.tcm.2008.01.002. PMID: 18436145.

10. Nakaya H. 2014; SCN5A mutations associated with overlap phenotype of long QT syndrome type 3 and Brugada syndrome. Circ J. 78:1061–1062. DOI: 10.1253/circj.CJ-14-0319. PMID: 24694743.

11. Chae H, Kim J, Lee GD, Jang W, Park J, Jekarl DW, Oh YS, Kim M, Kim Y. 2017; Considerations when using next-generation sequencing for genetic diagnosis of long-QT syndrome in the clinical testing laboratory. Clin Chim Acta. 464:128–135. DOI: 10.1016/j.cca.2016.11.013. PMID: 27871843.

12. O'Malley HA, Isom LL. 2015; Sodium channel β subunits: emerging targets in channelopathies. Annu Rev Physiol. 77:481–504. DOI: 10.1146/annurev-physiol-021014-071846. PMID: 25668026. PMCID: PMC4817109.

13. Gaborit N, Le Bouter S, Szuts V, Varro A, Escande D, Nattel S, Demolombe S. 2007; Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. J Physiol. 582:675–693. DOI: 10.1113/jphysiol.2006.126714. PMID: 17478540. PMCID: PMC2075332.

14. Makita N, Bennett PB Jr, George AL Jr. 1994; Voltage-gated Na+ channel beta 1 subunit mRNA expressed in adult human skeletal muscle, heart, and brain is encoded by a single gene. J Biol Chem. 269:7571–7578. DOI: 10.1016/S0021-9258(17)37325-8. PMID: 8125980.

15. Calhoun JD, Isom LL. 2014; The role of non-pore-forming β subunits in physiology and pathophysiology of voltage-gated sodium channels. Handb Exp Pharmacol. 221:51–89. DOI: 10.1007/978-3-642-41588-3_4. PMID: 24737232.

16. Daimi H, Lozano-Velasco E, Aranega A, Franco D. 2022; Genomic and non-genomic regulatory mechanisms of the cardiac sodium channel in cardiac arrhythmias. Int J Mol Sci. 23:1381. DOI: 10.3390/ijms23031381. PMID: 35163304. PMCID: PMC8835759.

17. Abriel H, Rougier JS, Jalife J. 2015; Ion channel macromolecular complexes in cardiomyocytes: roles in sudden cardiac death. Circ Res. 116:1971–1988. DOI: 10.1161/CIRCRESAHA.116.305017. PMID: 26044251. PMCID: PMC4471480.

18. Clatot J, Neyroud N, Cox R, Souil C, Huang J, Guicheney P, Antzelevitch C. 2020; Inter-regulation of K_v4.3 and voltage-gated sodium channels underlies predisposition to cardiac and neuronal channelopathies. Int J Mol Sci. 21:5057. DOI: 10.3390/ijms21145057. PMID: 32709127. PMCID: PMC7404392.

19. Wan X, Wang Q, Kirsch GE. 2000; Functional suppression of sodium channels by beta(1)-subunits as a molecular mechanism of idiopathic ventricular fibrillation. J Mol Cell Cardiol. 32:1873–1884. DOI: 10.1006/jmcc.2000.1223. PMID: 11013131.

20. Zimmer T, Surber R. 2008; SCN5A channelopathies--an update on mutations and mechanisms. Prog Biophys Mol Biol. 98:120–136. DOI: 10.1016/j.pbiomolbio.2008.10.005. PMID: 19027780.

21. Han D, Tan H, Sun C, Li G. 2018; Dysfunctional Nav1.5 channels due to SCN5A mutations. Exp Biol Med (Maywood). 243:852–863. DOI: 10.1177/1535370218777972. PMID: 29806494. PMCID: PMC6022915.