Published online before print December 6, 2001, doi: 10.1161/hh0102.102977
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© 2002 American Heart Association, Inc.
UltraRapid Communications |
Expression and Intracellular Localization of an SCN5A Double Mutant R1232W/T1620M Implicated in Brugada Syndrome
From the Department of Medicine, Laval University and Québec Heart Institute, Laval Hospital Research Center, Sainte-Foy, Québec, Canada.
Correspondence to Dr M. Chahine, PhD, Laval Hospital Research Center, 2725 Chemin Sainte-Foy, Sainte-Foy, Québec, Canada G1V 4G5. E-mail Mohamed.Chahine{at}phc.ulaval.ca
Abstract
Brugada syndrome is an inherited cardiac disorder caused by mutations in the cardiac sodium channel gene, SCN5A, that leads to ventricular fibrillation and sudden death. This study reports the changes in functional expression and cellular localization of an SCN5A double mutant (R1232W/T1620M) recently discovered in patients with Brugada syndrome. Mutant and wild-type (WT) human heart sodium channels (hNav1.5) were expressed in tsA201 cells in the presence of the ß1-auxiliary subunit. Patch-clamp experiments in whole-cell configuration were conducted to assess functional expression. Immunohistochemistry and confocal microscopy were used to determine the spatial distribution of either WT or mutant cardiac sodium channels. The results show an abolition of functional sodium channel expression of the hNav1.5/R1232W/T1620M mutant in the tsA201 cells. A conservative positively charged mutant, hNav1.5/R1232K/T1620M, produced functional channels. Immunofluorescent staining showed that the FLAG-tagged hNav1.5/WT transfected into tsA201 cells was localized on the cell surface, whereas the FLAG-tagged hNav1.5/R1232W/T1620M mutant was colocalized with calnexin within the endoplasmic reticulum (ER). These results indicate that a positively charged arginine or lysine residue at position 1232 in the double mutant is required for the proper transport and functional expression of the hNav1.5 protein. These results support the concept that loss of function of the cardiac Na+ channel is responsible for the Brugada syndrome. The full text of this article is available at http://www.circresaha.org.
Key Words: sodium channels • cardiac arrhythmias • protein trafficking • Brugada syndrome • ion channels
Brugada syndrome is an inherited primary cardiac disease that causes ventricular fibrillation and sudden death. It is characterized by ST-segment elevation of the right precordial ECG leads and a pseudo right bundle branch block that leads to ventricular tachycardia (VT) and ventricular fibrillation (VF).1,2 So far, mutations in only one gene, the SCN5A gene that encodes for the 
-subunit of the cardiac voltage-gated sodium channel (hNav1.5), have been identified as causing this syndrome, despite that this gene is responsible for approximately 20% of cases of Brugada syndrome.3
To date, 9 mutations of the SCN5A gene in patients with Brugada syndrome have been reported and biophysically characterized.4–8 Of these 9 mutations, 3 are shown to affect expression, whereas the others disrupt the function of the cardiac sodium channels. For instance, a missense mutation has been reported to abrogate translation, whereas another frameshift mutation that produces an aberrant protein has been reported to completely abolish sodium channel expression.5 Other mutations reduce expression levels of the human heart sodium channel9,10 or increase its inactivation kinetics.6,8 Recently, we reported that the absence of function of a Brugada mutant (R1432G) is due to disruption of protein trafficking toward the plasma membrane.11 When related to SCN5A gene, the loss of function of cardiac sodium channels, either by reducing expression levels or by increasing its inactivation kinetics, is the main pathological origin of the Brugada syndrome.7,12,13
Several recent studies have characterized a threonine-to-methionine mutation at position 1620 (T1620M) in SCN5A, found in Brugada syndrome patients.5,10,13–16 This mutation is located in the S3-S4 extracellular loop in domain IV of the hNav1.5. However, this mutation is also found in the presence of second mutation (R1232W) on the same allele of all affected individuals and located on S1-S2 extracellular loop in domain III.5
In this article, we report that the absence of function of hNav1.5/R1232W/T1620M double mutant is due to disruption of protein trafficking toward the plasma membrane. This protein trafficking defect is the main cause of Brugada syndrome phenotype in patients carrying the double mutation. Therefore, the genotyping procedure presently used to diagnose Brugada syndrome in the original family should include both mutations.
Materials and Methods
Generation of Expression Vectors
Mutants hNav1.5/T1620M, hNav1.5/R1232W, hNav1.5/R1232W/T1620M, hNav1.5/R1232K/T1620M, hNav1.5/R1232M/T1620M-FLAG, hNav1.5/WT-FLAG, and the ß1-subunit–c-myc were generated using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The tag epitope (FLAG) was inserted in the S5-S6 extracellular loop of domain I in both hNav1.5/WT and hNav1.5/R1232W/T1620M.11 In the ß1-subunit, c-myc epitope was introduced in the intracellular C-terminal side.11 The human sodium channel ß1-subunit and CD8 were constructed in pIRES bicistronic vector (pCD8-IRES-ß1).
All constructs were purified using Qiagen columns (Qiagen Inc), and the cDNA were sequenced to confirm the presence of the mutations and to ensure that no random mutations were introduced.
Transient Transfections of the tsA201 Cell Line
The tsA201 human cells were grown and transiently transfected using the calcium phosphate method previously described.7 Cells were grown at 37°C. After transfection, cells were maintained at 37°C or reduced to 22°C for a further period in culture, as noted. To better identify transfected cells for patch-clamp analysis, 10 µg of pCD8-IRES-ß1 plasmid was cotransfected with 10 µg of either the wild-type or the mutant hNav1.5 sodium channel cDNAs. For coexpression of wild-type with mutant channels, 2.5 µg of each sodium channel DNA in the presence of 10 µg pCD8-IRES-ß1 were used. Two to three days after transfection cells were incubated for 2 minutes in the extracelular solution containing anti–CD8-a coated beads (Dynabeads M-450 CD8-a) (Dynal AS).17
Cardiac Myocytes Isolation and Transfection
Myocytes were isolated from the ventricular cardiac tissue of 3-day-old Sprague Dawley rats (Charles River Canada, St-Constant, Quebec) using collagenase in calcium-magnesium–free Hanks buffer (Sigma). We essentially followed the isolation procedure described by Schanne et al.18 Cells were then cultured in DMEM milieu for 48 hours before transfection. The procedure used for cardiomyocyte transfection was as for tsA201 cells.
Solutions and Reagents
For whole-cell recording, the patch pipette contained (in mmol/L) 35 NaCl, 105 CsF, 10 EGTA, and 10 Cs-HEPES (pH 7.4). The extracellular solution contained (in mmol/L) 150 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 10 Na-HEPES (pH 7.4).
4-Phenylbutyrate (Triple Crown America, Inc) was used at 1 mmol/L concentration on cells and incubated at 37°C for 48 hours. Glycerol (Sigma) was used at 5% on cells incubated at 37°C for 24 hours.
Patch-Clamp Method
Macroscopic sodium currents from transfected cells were recorded using the whole-cell configuration of the patch-clamp technique as previously described.7
Immunocytochemistry
Transfected tsA201 cells or cardiac myocytes were first fixed using a 1:3 acetone/methanol solution for 20 minutes then permeabilized using 0.1% Triton into 1 mmol/L PBS-0.5% BSA solution before they were incubated with antibodies. The mouse anti-FLAG M2 primary antibody (1:4000) used against the FLAG-tagged -subunit of the sodium channel was purchased from Stratagene. The secondary antibody was a conjugated AffiniPure goat anti-mouse (1:4000) purchased from Molecular Probes (Eugene, Oreg, USA). Rabbit anti-calnexin polyclonal antibody (1:2500) was used for ER labeling from StressGen Biotechnologies Corp (Victoria, British Columbia, Canada).
Confocal Laser Microscopy
Fluorescent probe–labeled tsA201 cells or cardiac myocytes were viewed using a confocal imaging system (Bio-Rad MRC-1024) equipped with a Krypton-argon laser beam and mounted on a Zeiss microscope. A 60x-oil immersion objective with a 1.4 numerical aperture was used. Confocal settings were as follows: 1 mW laser power, zoom 1.2, 1 second per scan, Kalman filter, and 4 frames per image. The photomultiplier gain was set to maximum, and the confocal aperture was adjusted for maximum resolution.
Statistical Analysis
Data are expressed as mean±SEM (standard error of the mean). When indicated, t test analysis was performed using statistical software in SigmaPlot (Jandel Scientific Software). Differences were considered to be statistically significant with a value of P<0.05.
Results
Biophysical Properties of the R1232W, T1620M, and R1232W/T1620M Mutants
The SCN5A mutations (T1620M and R1232W) were first identified in patients with idiopathic ventricular fibrillation, known now as the Brugada syndrome.5 These mutations were expressed and biophysically characterized in the Xenopus oocyte expression system.5 The R1232W was reported to be a benign polymorphism because no changes in gating properties of sodium currents were observed.5 However, the T1620M mutation was found to change gating properties of sodium currents and was therefore thought to be the cause of Brugada syndrome phenotype. In the present study, these mutants (with single and double mutants) were reproduced in vitro using site-directed mutagenesis and characterized in mammalian cells in the presence of the ß1-subunit.
Sodium currents were recorded from cells transfected with WT, R1232W, T1620M, R1232W/T1620M, or R1232K/T1620M sodium channels and cotransfected with the ß1-subunit (Figure 1). The R1232W mutant resulted in a small shift in channel availability (-5 mV) compared with WT (Table). The expression levels of R1232W mutant were not affected (Figure 1B). However, as we reported earlier, the T1620M mutation resulted in changes in gating properties of voltage-gated sodium channels.13 A slow recovery from inactivation, normal current decay kinetics, and no shift in steady-state activation but a 9-mV shift in steady-state inactivation curve were observed (Table). Under identical conditions, the double mutant hNav1.5/R1232W/T1620M containing both amino acid substitutions produced no sodium currents (Figure 1D).
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To further characterize the origin of the loss of sodium currents, temperature-dependence of channel expression was also assessed. tsA201 cells were incubated at a lower temperature, 22°C for 36 to 48 hours before current recording, however no current could be recorded (Figure 1E).
We further characterized the importance of the positively charged arginine at position 1232 in the hNav1.5 sodium channel function by substituting the R1232 with lysine in the presence of T1620M mutation. Macroscopic sodium currents were recorded from cells expressing the hNav1.5/R1232K/T1620M mutant (Figure 1F). Steady-state activation and inactivation parameters of hNav1.5/R1232K/T1620M were similar to the T1620M mutant channel (Table).
Although the R1232W/T1620M double mutant did not express functional Na+ channels in tsA201 human cells, this mutant produced large currents when expressed in Xenopus oocytes (Data not shown). The detailed biophysical analysis of this mutant channel expressed in Xenopus oocytes was not carried out in the present study because 2 reports have already examined gating differences between wild-type, T1620M, and R1232W/T1620M mutant channels in this expression system.5,19
As illustrated in Figure 2, cotransfection with the same concentration of DNA encoding either for hNav1.5/WT or hNav1.5/WT+hNav1.5/R1232W/T1620M channels resulted in a 50% reduction in current density when mutant channels are present, whereas the lysine mutant hNav1.5/R1232K/T1620M generated similar current density compared with wild-type alone (-6541±987 pA/pF [n=6] for hNav1.5/WT; -3064±892 pA/pF [n=5] for hNav1.5/R1232W/T1620M+hNav1.5/WT; -5594±1084 pA/pF for hNav1.5/R1232K/T1620M).
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Confocal Laser Microscopy Study
Tagged hNav1.5 and ß1-subunit were expressed in tsA201 cells and in cardiac myocytes to assess the cellular localization of sodium channel proteins. Cardiac myocytes and tsA201 cells were transfected with tagged channel subunits to study trafficking of WT and mutant channels. In hNav1.5, the FLAG tag epitope was inserted in the S5-S6 extracellular loop of domain I, and in the ß1-subunit, the c-myc tag was introduced in the intracellular C-terminal side. Immunofluorescence staining shows that in tsA201 cells transfected with the hNav1.5/WT, the channel was localized at the cell surface in the presence of the ß1-subunit (Figure 3).
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In contrast, in cells transfected with the hNav1.5/R1232W/T1620M double mutant, immunofluorescence was restricted to the cytoplasmic region of the cell and was not found on the cell surface (Figure 3B). We also assessed the intracellular localization of the hNav1.5/R1232W/T1620M double mutant in the transfected tsA201 cells using the anti-calnexin antibody. Calnexin is a chaperon protein present in the endoplasmic reticulum (ER).20 Results of the colabeling studies show that both hNav1.5/R1232W/T1620M double mutant and calnexin were colocalized in the ER (Figures 3C and 3D).
It is noteworthy that when the double mutant and the ß1-subunit were cotransfected they were localized in distinct intracellular locations (Figures 3E, 3F, and 3G).
A similar pattern of colocalization was found in transfected newborn cardiac myocytes (Figure 4). Immunostaining of calnexin was localized in the ER (Figure 4A) and FLAG-tagged hNav1.5/WT was found in peripheral localizations (Figure 4B). Figure 4C represents the superimposition of both FLAG-tagged hNav1.5/WT (green) and calnexin (red). The hNav1.5/R1232W/T1620M double mutant (Figure 4D) and calnexin (Figure 4E) were colocalized within the ER (Figure 4F).
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Discussion
The purpose of this study was to characterize mutations found in patients with Brugada syndrome. The double mutation R1232W/T1620M is found on the same allele in patients with Brugada syndrome,5 suggesting that the presence of both mutations is responsible for the clinical phenotypes. Previous studies investigated the biophysical characterization of the T1620M single mutation. A temperature-dependent effect of the T1620M mutant was found, indicating that T1620M current decay kinetics were faster when compared with the wild-type at 32°C.14 These findings were later contradicted by Wan et al,16 where they reported a reduction in expression of the double mutation. An enhanced intermediate sodium channel inactivation10 and an alteration of mutant (T1620M) expression with the ß1-subunit were also suggested as possible molecular mechanisms for Brugada syndrome.15 We have reported that the T1620M mutation exhibited different phenotypes when expressed in Xenopus oocytes and in mammalian cells.13
Whereas the single mutants (R1232W and T1620M) were efficiently expressed in tsA201 cells, the double mutant R1232W/T1620M exhibited no functional channels. This resulted in a total absence of detectable sodium currents in transfected cells. We demonstrated earlier that the presence of a positive charge at position 1432 was essential for the proper trafficking of hNav1.5 sodium channel protein.11 To confirm the role of the positive charge at position 1232, the R1232 was replaced by lysine, another positive charge residue, along with T1620M substitution. Results show that the R1232K/T1620M mutant was functional, suggesting again that the presence of a positive charge at 1232 position is required for normal trafficking of sodium channel protein to the cell surface.
In contrast to the findings of Wan et al,16 the presence of the ß1-subunit did not influence the expression of the double mutant. We found no expression of the double mutant with or without the ß1-subunit. Thus the -ß1 interaction is not the cause of loss of expression observed in our study. The precise reason for this discrepancy is not clear.
It has been demonstrated that 4-phenylbutyric acid rescues the mutant CFTR channel trafficking defect by regulating the Hsc70 heat-shock protein.21 We tested the effect of 4-phenylbutyric acid on cells transfected with the hNav1.5/R1232W/T1620M mutant to determine whether the Hsc70 trafficking protein was involved. Cells were incubated for 48 hours after transfection in the presence of 1 mmol/L of 4-phenylbutyric acid at 37°C; however, no sodium current could be recorded from tsA201-transfected cells (data not shown). This suggested that Hsc70 was not involved in the retention of the mutated sodium channel protein within the ER. Lowering temperature from 37°C to 26°C has been found to restore HERG potassium channels in long QT syndrome.22 In our study, temperature-dependence of the protein processing was also tested. tsA201-transfected cells were incubated at 37°C and at 22°C for 48 hours after transfection. The absence of sodium currents in both cases ruled out the role of temperature-sensitive heat-shock chaperon protein in the processing of our mutant sodium channels. Glycerol was shown to correct defective trafficking of HERG mutant protein.23 No sodium current could be recorded from cells transfected with the double mutant when cells were incubated for 24 hours in the presence of 5% glycerol.
Immunostaining and laser confocal microscopy of cells transfected with tagged channel subunits showed that the mutant protein (hNav1.5/R1232W/T1620M) was retained within the cell. Our data also show that the hNav1.5/R1232W/T1620M double mutant is colocalized with the ER-specific chaperon protein (calnexin).20
This pattern was also observed when cardiac myocytes isolated from newborn rats were transiently transfected with hNav1.5/R1232W/T1620M double mutant but not with hNav1.5/WT. More studies are required to understand interactions between sodium channel trafficking and chaperon proteins. The reason for loss of cell surface localization of the double mutant channel is not well understood; however, one can speculate that the misfolding of mutant protein could be the cause of this defect. We propose that the individual mutations may independently induce a conformational change and, therefore, cause a cumulative misfolding on the channel protein. When cotransfected with the double mutant, the ß1-subunit was localized in a different region, most probably the Golgi, because our results suggest that the double is retained in the ER.
To mimic the dominant nature of the disease, we coexpressed the wild-type and mutant sodium channels using equal amounts of cDNAs. Our data document a 50% reduction in sodium current compared with cells expressing wild-type channels alone. It is hypothesized that reduction in sodium current will favor repolarization caused by potassium currents (Ito) and may lead to heterogeneity of repolarization in the right ventricle where Ito is more prevalent.24 In such a case, ST-segment of the surface ECG will be elevated on the right precordial leads V1 to V3 and will contribute to VT and VF.
It is noteworthy that the double mutant was successfully expressed in Xenopus oocytes (data not shown). This highlights again differences in voltage-gated sodium channel protein trafficking between this expression system and the mammalian expression system13,25,26 and emphasizes the importance of the characterization of human mutations in a human expression system.
In conclusion, our results show that the hNav1.5/R1232W/T1620M double mutant found in patients with Brugada syndrome is implicated in the trafficking of the cardiac sodium channel. Our data suggest that the retention of mutant channels within the ER is due to the absence of a positive charge located at position 1232. This study clarifies the pathophysiology of the R1232W/T1620M double mutant. We propose that the absence of a functional cardiac sodium channel is the main cause of the Brugada syndrome in carrier patients.
Acknowledgments
This study was supported by the Heart and Stroke Foundation of Québec, the Canadian Institutes of Health Research MT-13181, and by Fonds de la recherche en santé du Québec. Dr M. Chahine is an Edwards Senior investigator (Joseph C. Edwards Foundation). We thank Dr John G. Kingma for his comments on the manuscript.
Received October 10, 2001; revision received November 8, 2001; accepted November 20, 2001.
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