- Open Access
Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations
© Hsueh et al; licensee BioMed Central Ltd. 2009
- Received: 06 November 2008
- Accepted: 20 February 2009
- Published: 20 February 2009
The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively), and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV). In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.
- HEK293T Cell
- Sodium Current
- Brugada Syndrome
- Slow Inactivation
- Steady State Inactivation
SCN5A encodes the alpha subunit of human cardiac sodium channel, which is responsible for the generation of cardiac action potential and for rapid impulse conduction through the myocardium. Mutations in SCN5A cause inherited arrthymia syndrome such as Long QT syndrome (LQT3), Brugada syndrome, isolated conduction disease, atrial stanstill, congenital sick sinus syndrome or sudden infant death syndrome [2, 3]. Chen et al. first reported in 1998 that loss of function mutations of SCN5A accounts for the most well-known genetic basis for Brugada syndrom. However, for the clinically diagnosed cases, only no more than 20% carry SCN5A mutations. Mutations in other genes that cause Brugada syndrome have been reported. These genes include glycerol-3-phosphate dehydrogenase 1-like gene (GPD1L), the alpha subunit of the L-type calcium channel (CACNA1C), the beta subunit of the L-type calcium channel (CACNB2b), and the sodium channel beta subunit (SCN1B) [6–8]. However, SCN5A is so far still the most often reported gene causing Brugada syndrome. Altered electrophysiology, trafficking, expression level, or interaction with its intracellular components all has been reported to account for the mechanisms contribute to loss of function of SCN5A [9–12]. In this study, we investigated on three SCN5A mutation identified in patients with Brugada syndrome in Taiwan and tried to identify the underlying mechanism of three mutations that contribute to Brugada syndrome.
Cloning of SCN5A and SCN1B
Total RNA was extracted from human heart tissue using trizol (Invitrogen, USA) according to manufacturer's protocol. Complimentary DNA was synthesized using 200 units of Superscript III reverse transcriptase (Invitrogen, USA) at 52°C for 50 min in the presence of 5 μg of total RNA, 0.5 μg of oligo-dT primers, 0.004 mM DTT, 5% DMSO and 0.2 mM dNTPs, and the reaction product was used as the template in subsequent polymerase chain reaction(PCR). The PCR product for SCN1B was first cloned into pAAV-IRES-hrGFP (Stratagene, USA) with BamH I/Xho I and subcloned into the Bgl II recognition sequence of pBudCE4.1 (Invitrogen) with BamH I/Bgl II. The subclone procedure allowed dicistronic expression of SCN1B and humanized Renilla reniformis green fluorescent protein (hrGFP) under the control of EF-1α promoter. The PCR product of SCN5A was cloned into the pBudCE4.1 containing SCN1B with Hind III/Xba I under the control of CMV promoter. Besides, a myc epitope EQKLISEEDL was introduced in frame at the N terminus of SCN5A. The base sequence of the SCN5A and SCN1B clone were analyzed and were identical to the published SCN5A (hH1, NM_198056) and SCN1B (NM_199037) sequence.
Site directed mutagenesis
The p.I848fs, p.R965C, and p.1876insM mutants were generated using the QuickChange Site-Directed Mutagenesis system (Stratagene). All constructs were sequenced to verify the mutation and to rule out possible PCR errors.
Culture and transfection of HEK293T cells
HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics at 37°C and 5% CO2. Jetpei (Polyplus) was used as the reagent for transient transfection. Briefly, 3 × 105 HEK293T cells were seeded into a 35 mm dish the day before transfection. Onto the cell monolayer were added 3 μg plasmid and 6 μl Jetpei in 200 μgl sodium chloride solution (150 mM). The cells were harvested by PBS containing 1% EDTA for patch clamp or for western blot 48–56 hrs after transfection.
Patch clamp and data analysis
Cells were incubated in bath solution containing (mM): NaCl 145, KCl 4, CaCl 1.8, MgCl 1, HEPES-NaOH pH 7.35. Expressed currents were recorded by whole-cell patch-clamp technique using a patch-clamp amplifier (Dagan 8900). The pipette solution contained (mM) NaF 10, CsF 110, CsCl 20, EGTA 10 and HEPES 10 (pH 7.35 with CsOH). Pipettes were made from borosilicate glass capillaries and had tip resistances between 1.5 and 2.5 MÙ when filled with the pipette solution. All of the electrical recordings were performed at room temperature (24–26°C). Data acquisition was performed through a DigiData 1200 amplifier controlled by pClamp 6.0, and the results were analyzed using Clampfit 9.0.
To measure the cell capacitance, a step voltage (from -80 mV to -70 mV) was applied to the cell and accessed the area under the capacity transient. The cell capacitance was obtained by dividing the area under the capacity transient with the factor 10. The plots of voltage dependent steady state activation and inactivation were fitted by Boltzmann equation: Y = 1/[1+exp(V -Vm)/k], where Vm is the voltage at which sodium current is half-maximally activated, and k was the slope factor. Time constants of inactivation were obtained by fitting the decaying phase of current trace with one exponential equation: Y = A*exp(-t/τ) + C. To analyze the kinetics of recovery from inactivation, two-exponential equation was as in the format: Y = A*[1-exp(-t/τ f)]+(1-A)*[1-exp(-t/τ s)].
Immunocytochemistry and Confocal imaging
HEK293T cells were cultivated on chamber slips (UNUC) and transiently transfected with WT, p.I848fs, p.R965C, or p.1876insM. Forty-eight hours after transfection, the culture medium were removed and cells were washed with ice-cold PBS. The cells were then fixed by incubation in 4% paraformaldehyde at room temperature for 30 min and permeabilized with PBS containing 0.5% tween 20 and 10% BSA for another 30 min. Before comfocal imaging, cells were incubated in PBS containing primary antibody (anti-myc 1:500, Upstate) overnight at 4°C and secondary antibody anti-mouse IgG coujugated with Cy3 (1:2000, Sigma) for 30 min. Confocal images were obtained and analyzed using a Leica TCS SP5 Spectral Confocal System.
Surface biotinylation reaction
Sulfo-NHS-LC-Biotin (PIERCE) was used as the reagent for labeling of cell surface proteins. Briefly, the transfected cells were washed and harvested using 1%EDTA (prepared in PBS). The cell pellets were resuspended in PBS containing Sulfo-NHS-LC-Biotin (2 mg/ml) and rotated at 4°C for 30 min. The biotinylation reaction was blocked by adding equal volume of 0.1 M glycine (in PBS) and rotated for mixing for 20 min at 4°C. The cells were then centrifuged and wash 3 times with PBS and subjected to protein extraction and Western blotting.
The cells for protein extraction were collected 48 to 56 hours after transfection by centrifuging at 500 g for 5 min. The pellet was then incubated in lysis buffer (1% Triton-X 100 pH 8.0, 50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, 0.02% sodium azide, 1 mM PMSF, 2 μg/ml leupeptin, and 5 μg/ml apotinin) for 30 min, and centrifuged at 15000 g for 15 min.
For analysis of biotinylated protein, the extracted proteins were mixed with anti-myc antibody (Upstate) and protein A/G plus (Santa Cruz) for 4 hrs, washed 4 times with lysis buffer and the immunoprecipitated proteins were released from the beads by heating at 95°C for 5 min in 5× sample buffer. The collected proteins were subjected to SDS-PAGE, transferred to PVDF membrane, and detected by Western blotting analysis using HRP conjugated streptavidin (PIERCE).
Data management and statistical analysis
Data of patch clamp were ananlyzed using clampfit 9.0, and photoshop 8.0 was used for confocal image processing. Prism 4.0 was used for figure plotting, curve fitting and statistical calculation. Data were presented as mean ± standard error of the mean (SEM). Statistical comparisons were compared to WT using unpaired Student's t test for current density, Vm, slop factor, and time constants. Multiple group comparison was made using one way ANOVA followed by Tukey test. P values less than 0.05 were taken as statistically significant.
Genetic analysis and the electrocardiographic (ECG) characteristics
Sodium current elicited by WT and mutant cardiac sodium channels
Voltage dependent steady state activation and steady state inactivation
Time constants of fast inactivation
On stimulation, sodium channel open and inactivated rapidly. Fast inactivation was accessed by analysis of time constant of fast inactivation. By fitting the decaying phase of sodium current, we found that the time constants of inactivation for p.1876insM were significant larger than WT (Figure 3B). This suggested that p.1876insM inactivated slower than WT during depolarizing stimulation.
Recovery from inactivation and development of slow inactivation
Cell surface protein biotinylation and Western blotting
Intracellular trafficking analysis by confocal imaging
We characterized three mutations in SCN5A among Taiwanese patients with Brugada syndrome using patch clamp technique, western blotting and confocal imaging. Western blotting and confocal imaging showed that three mutants were trafficking-competent. However, their electrophysiological properties were impaired when compared with WT. p.I848fs elicited no sodium current. p.R965C produced sodium channel with impaired steady state inactivation, recovery from inactivation and slow inactivation while p.1876insM generated sodium channel with altered kinetics of activation, inactivation, and recovery from inactivation. We recognized that a decreased of sodium current might contribute to Brugada syndrome because all the three mutations resulted in a decrease of sodium current. However, the degree of sodium channel decrease does not seem to correlate with conduction time such as PR interval. Some other factors might compensate or play unknown roles in the clinical presentation.
SCN5A encodes the alpha subunit of cardiac sodium channel, which consists four homologous domains, and each domain contains six alpha-helical transmembrane repeats. The frame shift of the p.I848fs mutant located at the fifth transmembrane segment of domain 2 (DII/S5) of cardiac sodium channel. Because it produces a protein with a truncation of more than a half of the WT cardiac sodium channel protein, the observation that it elicited no current was reasonable. In constancy with our founding, Shin et al. reported a mutation, W1119X, which lacked even less amino acids than p.I848fs, failed to generate any current. Moreover, by western blotting and confocal imaging analysis, the presence of p.I848fs at cytoplasmic membrane suggested that the remained amino acid residues in I848fs were sufficient for the processing and trafficking of cardiac sodium channel.
The substituted amino acid p.R965C located at the intracellular loop between domain II and domain III of SCN5A protein (DII-DIII). Mutations located at this region had been reported http://www.fsm.it/cardmoc/, but few had been characterized by functional studies. Arginine at position 965, together with nearby amino acids, formed a specialized structure with putative amphiphilic helix carrying regularly arranged positive charges which resembling an S4 segment of voltage-gated ion channels. The sequence here was unique to cardiac sodium channel. Camacho et al. had reported a splice variant lacking this sequence and showed that was associated with altered steady state activation, and inactivation. Besides, Camacho et al. also pointed out that three positively charged arginine in this region were involved in current density. Arginine at 965 position located at one of them (the other two were at 968 and 971). The substitution of p.R965C from the positively charged aginine to a neutral cysteine might influence channel gating properties. In this study we observed a left shift in inactivation curve, and altered recovery from inactivation. The current density was not significantly changed, although it seemed to be smaller. Besides, the altered steady state inactivation might partly be due to impaired slow inactivation as we observed.
There had been many functional studies reported for the mutations in the carboxyl-terminal of cardiac sodium channel and implied its role in controlling channel inactivation [16–18]. As predicted by the amino acid segment, the carboxyl-terminal domain of cardiac sodium channel can to be divided into two parts. The proximal part was structured and forms six helices, whereas the distal part (about 100 amino acids) is unstructured. Loss of the predicted six helices greatly destabilizes inactivation while truncation of the unstructured part does not affect no channel gating. The six helices of the carboxyl -terminal might interact with the linker of domain III and domain IV during inactivation. Changes of amino acid at these regions influences inactivation and contributes to Brugada syndrome, LQT3 or both, such as del KPQ1507-1509 (DIII-DIV), E1784K (C-terminal) or 1795insD(C-terminal) [20–23]]. The insertion of the p.1876insM mutant located at the fifth helices of the carboxyl-terminal and this mutation was found to have an altered inactivation. This was in agreement with the previous reports [16–18]. Besides inactivation, mutations located at the carboxyl-terminal might affect other gating parameters [20–22, 24–26]. Rivolta et al. reported two mutations, Y1795H and Y1795C, contributing to Brugada syndrome and LQT3 respectively . These two mutations affected steady state inactivation, fast inactivation, current density, and were more prone to enter slow inactivation without affecting steady state activation and recovery from inactivation. Similar findings were also observed in the Brugada syndrome mutation, C1859S as reported by Petitprez et al. In this study, we found that the p.1876insM mutant at the carboxyl-terminal affected more than these parameters. Change of steady state activation of p.1876insM was observed as Shirai et al. observed in T1620M and S1710L . This again proved the importance of carboxyl-terminal in regulation of cardiac sodium channel.
Some residues of cardiac sodium channel were important in regulation of the gating property of channel. Changes or lacks of these residues might therefore contribute to the alteration of electrophysiological property as we showed in this study. The three mutations accounted for Brugada syndrome all had electrophysiological alteration and contributed to loss of function of cardiac sodium channel. A more detailed screening of relationship between structure and channel gating might be worthwhile since similar results were observed for mutations located at different positions.
- Balser JR: The cardiac sodium channel: gating function and molecular pharmacology. Journal of molecular and cellular cardiology. 2001, 33: 599-613. 10.1006/jmcc.2000.1346.View ArticlePubMedGoogle Scholar
- Tan HL, Bezzina CR, Smits JPP, Verkerk AO, Wilde AAM: Genetic control of sodium channel function. Cardiovascular research. 2003, 57: 961-973. 10.1016/S0008-6363(02)00714-9.View ArticlePubMedGoogle Scholar
- George AL: Inherited disorders of voltage-gated sodium channels. J Clin Invest. 2005, 115: 1990-1999. 10.1172/JCI25505.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, Potenza D, Moya A, Borggrefe M, Breithardt G, Ortiz-Lopez R, Wang Z, Antzelevitch C, O'Brien RE, Schulze-Bahr E, Keating MT, Towbin JA, Q W: Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature. 1998, 392: 293-296. 10.1038/32675.View ArticlePubMedGoogle Scholar
- Rossenbacker T, Priori SG: The Brugada syndrome. Current opinion in cardiology. 2007, 22: 163-170. 10.1097/HCO.0b013e328112f345.View ArticlePubMedGoogle Scholar
- Watanabe H, Koopmann TT, Le Scouarnec S, Yang T, Ingram CR, Schott JJ, Demolombe S, Probst V, Anselme F, Escande D, Wiesfeld AC, Pfeufer A, Kaab S, Wichmann HE, Hasdemir C, Aizawa Y, Wilde AA, Roden DM, Bezzina CR: Sodium channel beta1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans. The Journal of clinical investigation. 2008, 118: 2260-2268.PubMed CentralPubMedGoogle Scholar
- Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, Oliva A, Wollnik B, Gelber P, Bonaros EP, Burashnikov E, Wu Y, Sargent JD, Schickel S, Oberheiden R, Bhatia A, Hsu L-F, Haissaguerre M, Schimpf R, Borggrefe M, Wolpert C: Loss-of-Function Mutations in the Cardiac Calcium Channel Underlie a New Clinical Entity Characterized by ST-Segment Elevation, Short QT Intervals, and Sudden Cardiac Death. Circulation. 2007, 115: 442-449. 10.1161/CIRCULATIONAHA.106.668392.PubMed CentralView ArticlePubMedGoogle Scholar
- London B, Michalec M, Mehdi H, Zhu X, Kerchner L, Sanyal S, Viswanathan PC, Pfahnl AE, Shang LL, Madhusudanan M, Baty CJ, Lagana S, Aleong R, Gutmann R, Ackerman MJ, McNamara DM, Weiss R, Dudley SC: Mutation in Glycerol-3-Phosphate Dehydrogenase 1 Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias. Circulation. 2007, 116: 2260-2268. 10.1161/CIRCULATIONAHA.107.703330.PubMed CentralView ArticlePubMedGoogle Scholar
- Baroudi G, Acharfi S, Larouche C, Chahine M: Expression and intracellular localization of an SCN5A double mutant R1232W/T1620M implicated in Brugada syndrome. Circ Res. 2002, 90: E11-16. 10.1161/hh0102.102977.View ArticlePubMedGoogle Scholar
- Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG, Bennett V: Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101: 17533-17538. 10.1073/pnas.0403711101.PubMed CentralView ArticlePubMedGoogle Scholar
- Grant AO: Electrophysiological basis and genetics of Brugada syndrome. J Cardiovasc Electrophysiol. 2005, 16 (Suppl 1): S3-7. 10.1111/j.1540-8167.2005.50104.x.View ArticlePubMedGoogle Scholar
- Bezzina CR, Shimizu W, Yang P, Koopmann TT, Tanck MW, Miyamoto Y, Kamakura S, Roden DM, Wilde AA: Common sodium channel promoter haplotype in asian subjects underlies variability in cardiac conduction. Circulation. 2006, 113: 338-344. 10.1161/CIRCULATIONAHA.105.580811.View ArticlePubMedGoogle Scholar
- Priori SG, Napolitano C, Gasparini M, Pappone C, Della Bella P, Brignole M, Giordano U, Giovannini T, Menozzi C, Bloise R, Crotti L, Terreni L, Schwartz PJ: Clinical and Genetic Heterogeneity of Right Bundle Branch Block and ST-Segment Elevation Syndrome: A Prospective Evaluation of 52 Families. Circulation. 2000, 102: 2509-2515.View ArticlePubMedGoogle Scholar
- Shin DJ, Kim E, Park SB, Jang WC, Bae Y, Han J, Jang Y, Joung B, Lee MH, Kim SS, Huang H, Chahine M, Yoon SK: A novel mutation in the SCN5A gene is associated with Brugada syndrome. Life sciences. 2007, 80: 716-724. 10.1016/j.lfs.2006.10.025.View ArticlePubMedGoogle Scholar
- Camacho JA, Hensellek S, Rougier JS, Blechschmidt S, Abriel H, Benndorf K, Zimmer T: Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region. The Journal of biological chemistry. 2006, 281: 9498-9506. 10.1074/jbc.M509716200.View ArticlePubMedGoogle Scholar
- Motoike HK, Liu H, Glaaser IW, Yang A-S, Tateyama M, Kass RS: The Na+ Channel Inactivation Gate Is a Molecular Complex: A Novel Role of the COOH-terminal Domain. J Gen Physiol. 2004, 123: 155-165. 10.1085/jgp.200308929.PubMed CentralView ArticlePubMedGoogle Scholar
- Tateyama M, Liu H, Yang AS, Cormier JW, Kass RS: Structural Effects of an LQT-3 Mutation on Heart Na+ Channel Gating. Biophys J. 2004, 86: 1843-1851. 10.1016/S0006-3495(04)74251-4.PubMed CentralView ArticlePubMedGoogle Scholar
- Kass RS: Sodium Channel Inactivation in Heart: A Novel Role of the Carboxy-Terminal Domain. Journal of cardiovascular electrophysiology. 2006, 17: S21-S25. 10.1111/j.1540-8167.2006.00381.x.View ArticlePubMedGoogle Scholar
- Cormier JW, Rivolta I, Tateyama M, Yang AS, Kass RS: Secondary structure of the human cardiac Na+ channel C terminus: evidence for a role of helical structures in modulation of channel inactivation. The Journal of biological chemistry. 2002, 277: 9233-9241. 10.1074/jbc.M110204200.View ArticlePubMedGoogle Scholar
- Bezzina C, Veldkamp MW, Berg van Den MP, Postma AV, Rook MB, Viersma JW, van Langen IM, Tan-Sindhunata G, Bink-Boelkens MT, Hout van Der AH, Mannens MM, Wilde AA: A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res. 1999, 85: 1206-1213.View ArticlePubMedGoogle Scholar
- Wei J, Wang DW, Alings M, Fish F, Wathen M, Roden DM, George AL: Congenital long-QT syndrome caused by a novel mutation in a conserved acidic domain of the cardiac Na+ channel. Circulation. 1999, 99: 3165-317124.View ArticlePubMedGoogle Scholar
- Viswanathan PC, Bezzina CR, George AL, Roden DM, Wilde AA, Balser JR: Gating-dependent mechanisms for flecainide action in SCN5A-linked arrhythmia syndromes. Circulation. 2001, 104: 1200-1205. 10.1161/hc3501.093797.View ArticlePubMedGoogle Scholar
- Keller DI, Acharfi S, Delacretaz E, Benammar N, Rotter M, Pfammatter JP, Fressart V, Guicheney P, Chahine M: A novel mutation in SCN5A, delQKP 1507–1509 causing long QT syndrome: role of Q1507 residue in sodium channel inactivation. Journal of molecular and cellular cardiology. 2003, 35: 1513-1521. 10.1016/j.yjmcc.2003.08.007.View ArticlePubMedGoogle Scholar
- Rivolta I, Abriel H, Tateyama M, Liu H, Memmi M, Vardas P, Napolitano C, Priori SG, Kass RS: Inherited Brugada and long QT-3 syndrome mutations of a single residue of the cardiac sodium channel confer distinct channel and clinical phenotypes. The Journal of biological chemistry. 2001, 276: 30623-30630. 10.1074/jbc.M104471200.View ArticlePubMedGoogle Scholar
- Petitprez S, Jespersen T, Pruvot E, Keller DI, Corbaz C, Schlapfer J, Abriel H, Kucera JP: Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome. Cardiovascular research. 2008, 78: 494-504. 10.1093/cvr/cvn023.View ArticlePubMedGoogle Scholar
- Shirai N, Makita N, Sasaki K, Yokoi H, Sakuma I, Sakurada H, Akai J, Kimura A, Hiraoka M, Kitabatake A: A mutant cardiac sodium channel with multiple biophysical defects associated with overlapping clinical features of Brugada syndrome and cardiac conduction disease. Cardiovascular research. 2002, 53: 348-354. 10.1016/S0008-6363(01)00494-1.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.