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(Circulation Research. 2001;88:e78.)
© 2001 American Heart Association, Inc.

UltraRapid Communication

Novel Mechanism for Brugada Syndrome

Defective Surface Localization of an SCN5A Mutant (R1432G)

Ghayath Baroudi, Valerie Pouliot, Isabelle Denjoy, Pascale Guicheney, Alvin Shrier, Mohamed Chahine

From Laval University, Department of Medicine (G.B., V.P., M.C.) and Québec Heart Institute, Laval Hospital, Research Center (G.B., V.P., M.C.), Sainte-Foy, Québec, Canada; Service de Cardiologie, Hôpital Lariboisière (I.D.) and INSERM U523, Institut de Myologie, IFR "Cur, muscles et vaisseaux" No. 14, Groupe hospitalier Pitié-Salpêtrière (P.G.), Paris, France; and Department of Physiology, McGill University (A.S.), Montreal, Canada.

Correspondence to Dr M. Chahine, Laval Hospital Research Center, 2725 Chemin Sainte-Foy, Sainte-Foy, Québec, Canada G1V 4G5. E-mail Mohamed.Chahine{at}phc.ulaval.ca

Abstract

Abstract—The SCN5A gene encodes the subunit of the human heart sodium channel (hH1), which plays a critical role in cardiac excitability. Mutations of SCN5A underlie Brugada syndrome, an inherited disorder that leads to ventricular fibrillation and sudden death. This study describes changes in cellular localization and functional expression of hH1 in a naturally occurring SCN5A mutation (R1432G) reported for Brugada syndrome. Using patch-clamp experiments, we show that there is an abolition of functional hH1 expression in R1432G mutants expressed in human tsA201 cells but not in Xenopus oocytes. In tsA201 cells, a conservative positively charged mutant, R1432K, produced sodium currents with normal gating properties, whereas other mutations at this site abolished functional sodium channel expression. Immunofluorescent staining and confocal microscopy showed that the wild-type subunit expressed in tsA201 cells was localized to the cell surface, whereas the R1432G mutant was colocalized with calnexin within the endoplasmic reticulum. The ß₁ subunit was also localized to the cell surface in the presence of the subunit; however, in its absence, the ß₁ subunit was restricted to a perinuclear localization. These results demonstrate that the disruption of SCN5A cell-surface localization is one mechanism that can account for the loss of functional sodium channels in 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 is characterized by an elevated ST segment and a pseudo right bundle-branch block.^{1 2} To date, all patients reported with this syndrome exhibit defects in the SCN5A gene, shown in Figure 1, ^{3 4 5 6} which encodes the subunit of the cardiac voltage-gated sodium channel (hH1). Among the 9 mutations identified, a frameshift mutation was found to completely abolish cardiac sodium channel expression,⁴ whereas other mutants were associated with either reduced levels of channel expression^{7 8 9} or increased inactivation kinetics.^{5 10} We recently reported an arginine-to-glycine mutation at position 1432 (R1432G) in SCN5A in a patient with Brugada syndrome.⁶ This mutation, found in the extracellular loop between the pore region and the S6 transmembrane segment in domain III of the subunit, completely abolishes sodium current expression in tsA201 cells. In this study, we demonstrate that the mechanism underlying this abolition of channel function is a disruption in the localization of the subunit of hH1 to the plasma membrane.

View larger version (44K): [in this window] [in a new window]   Figure 1. The subunit of the SCN5A cardiac sodium channel is a transmembrane protein with four domains. Black circles (•) identify the Brugada syndrome mutations reported to date. The frameshift mutation consists of two nucleotides insertion (AA) in the splice-donor sequence of intron 7. The stop codon is produced by a deletion of one nucleotide (A) at position 1398. The R1432G, T1620 mol/L, R1512W, S1710L, A1924T, and L567Q represent missense mutations that cause Brugada syndrome. Note that the D1795ins causes both long-QT and Brugada syndrome.

Materials and Methods

Generation of Expression Vectors
Mutants hH1/R1432G, reverse mutant G1432R, hH1/R1432K, hH1/R1432C, hH1/R1432H, hH1/WT-FLAG, hH1/R1432G-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 to S6 extracellular loop of domain I in both hH1/WT and hH1/R1432G. In the ß₁ subunit, c-myc epitope was introduced in the intracellular C-terminal side. 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 ensure that other random mutations were not introduced.

Transfections of the tsA201 Cell Line
The tsA201 human cells were grown and transfected using the calcium phosphate method previously described.⁶ Cells were grown at 37°C. After transfection, cells were maintained at 37°C or reduced to either 22°C or 29°C for an additional 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 (WT) or the mutant hH1 sodium channel cDNAs. Two to three days after transfection, cells were incubated for 2 minutes in a medium containing beads coated with anti–CD8-a (Dynabeads M-450 CD8-a) (Dynal A.S.).¹¹

Solutions and Reagents
For whole-cell recording, the patch pipette contained, in mmol/L, NaCl 35, CsF 105, EGTA 10, and Cs-HEPES 10 (pH 7.4). The extracellular solution contained, in mmol/L, NaCl 150, KCl 2, CaCl₂ 1.5, MgCl₂ 1, glucose 10, and Na-HEPES 10 (pH 7.4). For experiments in which extracellular pH was altered, the following solution was used (in mmol/L): NaCl 150, KCl 2, CaCl₂ 1.5, MgCl₂ 1, glucose 10, and Na-HEPES 10 (pH 6.2). 4-Phenyl butyrate (Triple Crown America, Inc) was used at 1 mmol/L concentration on cells and incubated at 37°C for 48 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.¹²

Expression in Xenopus Oocytes
Xenopus oocytes were obtained and injected as previously reported.¹² The macroscopic sodium currents from cRNA-injected oocytes were measured by 2-microelectrode voltage clamp using an OC-725 Oocyte clamp (Warner Instruments). The Ringer solution bath contained, in mmol/L, NaCl 116, KCl 2, CaCl₂ 1.8, MgCl₂ 2, and Na-HEPES 5 (pH 7.6). Experiments were performed at room temperature (22°C to 23°C).

Immunocytochemistry
Transfected cells were permeabilized using 0.1% Triton into 1 mmol/L PBS-0.5% BSA solution before they were incubated with the antibodies. Cells were fixed using a 1:3 acetone/methanol solution for 20 minutes. The mouse anti-FLAG M2 primary antibody (1:4000) used against the FLAG-tagged subunit of the sodium channel was purchased from Stratagene (La Jolla, Calif). The secondary antibody was a conjugated AffiniPure goat anti-mouse (1:400) purchased from Molecular Probes (Eugene, Oreg). Primary antibody rabbit anti–c-myc (A-14) (1:1000) used against the c-myc–tagged ß₁ subunit was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). The secondary antibody (anti-rabbit) used in 1:100 dilution was from Molecular Probes (Eugene, Oreg). Rabbit Anti-Calnexin polyclonal antibody (1:2500) was used for endoplasmic reticulum (ER) labeling from StressGen Biotechnologies Corp (Victoria, British Columbia, Canada).

Confocal Laser Microscopy
Fluorescent probe–labeled tsA201 cells were viewed by a Bio-Rad MRC-1024 confocal imaging system equipped with a krypton-argon laser beam and mounted on a Zeiss microscope. A x60 oil objective with a 1.4 numerical aperture was used. Confocal settings were as follows: 1-mw laser power, 1.2 zoom, 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.

Results

Electrophysiological Properties of the R1432G Mutant
To verify the abolition of sodium current expression in the R1432G defect of SCN5A, we used human tsA201 cells transfected with the ß₁ subunit and WT (Figure 2A) or mutant hH1 sodium channels (Figure 2B). Cells expressing the R1432G mutant channels had no detectable sodium current, in contrast with the macroscopic sodium currents recorded with WT channels (Figures 2A and 2B). Sodium currents were also absent in R1432G-transfected cells incubated at 22°C for 36 to 48 hours before current recording, demonstrating that the expression defect is not temperature-dependent (Figure 2C).

View larger version (21K): [in this window] [in a new window]   Figure 2. Sodium current traces recorded from tsA201 cells expressing WT (A), the mutant R1432G incubated at 37°C for 36 to 48 hours after transfection (B), the mutant R1432G incubated at 29°C for 48 hours after transfection (C), and the reverse-mutant G1432R (D). Cells were cotransfected with the ß1 regulatory subunit. Currents were recorded from a holding potential of -140 mV stepped from -80 to +60 mV during 40 ms in 10-mV increments.

Figure 2D shows that a reverse mutant, G1432R, restored sodium current with normal kinetics and gating properties, indicating that the absence of current was attributable to the R1432G substitution. To additionally characterize the importance of the positively charged arginine at position 1432 in SCN5A, three other mutants (R1432C, R1432K, and R1432H) were constructed. The R1432K mutant was found to express macroscopic sodium currents (Figure 3A) that displayed voltage-dependent steady-state activation and inactivation properties that were indistinguishable from the WT channel (Figure 4A). The V_1/2 values for steady-state activation and inactivation curves for the R1432K mutant and the WT were similar: V_1/2^hH1/R1432K=-52.17±3.04 mV versus V_1/2^hH1/WT=-47.23±1.8 mV (n=5) for activation and V_1/2^hH1/R1432K=-92.24±1.4 mV versus V_1/2^hH1/WT=-92.47±1.13 mV (n=4) for inactivation. No significant changes were observed in the slope factors for either activation or inactivation curves (Figure 4A). Time constants of recovery from fast inactivation of R1432K and WT were also comparable (Figure 4B). Neither the R1432C (data not shown) nor the R1432H mutant expressed measurable sodium currents. Furthermore, in the case of the R1432H mutant, acidification of the medium to pH 6.2, which promotes histidine protonation, did not restore the sodium current (Figures 3B and 3C). This suggests that the R1432H residue is not accessible from the extracellular milieu.

View larger version (14K): [in this window] [in a new window]   Figure 3. Sodium current traces recorded from tsA201 cells expressing R1432K (A), R1432H in an extracellular bath at pH 7.4 (B), and the mutant R1432G in an extracellular bath at pH 6.2 (C). Currents were recorded from a holding potential of -140 mV stepped from -80 to +60 mV in 10-mV increments, each lasting 40 ms.

View larger version (16K): [in this window] [in a new window]   Figure 4. Voltage dependence of activation and inactivation and time course of recovery from fast inactivation. A, Overlap in the steady-state activation and inactivation curves for the WT and the R1432K-mutated cardiac sodium channel. Sodium currents were recorded from a holding potential of -140 mV stepped from -80 to +60 mV in 10-mV increments for activation and with +10-mV test pulse initiating from holding potentials stepped from -140 to -20 mV in 10-mV increment for inactivation. B, Overlap in time constants of the recovery from fast inactivation studied using two-pulse protocol of -20 mV at -120-mV holding potential. In both panels, white circles () correspond to the WT and black circles (•) correspond to the R1432K mutant.

Interestingly, when the R1432G mutant was expressed in Xenopus oocytes, sodium current was recorded with normal gating properties, showing that the loss of the positively charged residue is not the cause of disruption of channel function (Figure 5B).

View larger version (20K): [in this window] [in a new window]   Figure 5. Sodium current traces recorded from Xenopus oocytes expressing the WT sodium channel (A) and the R1432G mutant (B). Currents were recorded from a holding potential of -100 mV stepped from -80 to +20 mV in 10-mV increments. C, Current-voltage relationship of normalized sodium current peaks recorded from WT and R1432G mutant plotted against voltage.

To determine whether the Hsc70 trafficking protein is implicated in the absence of sodium currents in R1432G mutants, we exposed transfected cells to 4-phenyl butyrate. Cells incubated for 48 hours after transfection in the presence of 4-phenyl butyrate did not show measurable sodium currents (data not shown).

Confocal Laser Microscopy
To study cellular localization of WT and mutant channels, tsA201 cells were transfected with tagged channel subunits. The tagged constructs generated sodium currents with densities and biophysical properties similar to those of WT subunits (data not shown). Immunofluorescence staining shows that the hH1/WT-FLAG construct was localized to the cell membrane in either the presence or absence of the c-myc–tagged ß₁ subunit (Figures 6A and 6C). In contrast, the c-myc–tagged ß₁ subunit required coexpression of the subunit (Figure 6A) for cell-surface localization. When expressed in the absence of the subunit, the c-myc–tagged ß₁ subunit is localized as an agglomerate in the perinuclear region, suggesting that association with the subunit is a necessary step for normal localization to the cell-surface membrane.

View larger version (30K): [in this window] [in a new window]   Figure 6. Immunostaining of tsA201 cells expressing the hH1 cardiac sodium channels. A, Cell transfected with hH1/WT-FLAG shows peripheral localization of the channel. B, Cell transfected with ß1 subunit alone shows a perinuclear localization. C, Cell cotransfected with both hH1/WT-FLAG and ß1-c-myc shows also a peripheral localization of the channel. D and E, Cells transfected with the hH1/R1432G-FLAG mutant expressed alone and with the ß1-c-myc, respectively. The channel seems to be blocked within cytoplasmic vesicles. Transfected cells were immunostained with anti-FLAG (green) for the subunits and anti–c-myc for ß1 subunit (red). No detectable immunofluorescence staining was observed in nontransfected cells (data not shown).

Cell-surface localization was disrupted in cells transfected with the hH1/R1432G-FLAG mutant. Instead, immunofluorescent labeling was found within vesicles spread throughout the cytoplasm (Figures 6D and 6E). Furthermore, Figure 7 shows that the hH1/R1432G-FLAG mutant was colocalized with calnexin in the ER (Figure 7).

View larger version (25K): [in this window] [in a new window]   Figure 7. Immunostaining of tsA201 cells expressing the hH1 cardiac sodium channels. A, Cell transfected with hH1/R1432G-FLAG, labeled with the anti-FLAG (green), shows peripheral localization of the channel. B, Same cell shows the anticalnexin (red) present specifically in the ER. C, Superimposition of the green and red (orange), showing colocalization of hH1/WT-FLAG and the calnexine in ER vesicles.

Discussion

This study provides a mechanism to explain the pathophysiology of a SCN5A mutant (R1432G) previously reported to cause Brugada syndrome.⁶ In this study, we present an evaluation of the function of several mutant channels with substitutions at residue 1432 that is found in the S5 to S6 region of domain III, believed to be the pore of the cardiac sodium channel. Three mutants (R1432G, R1432C, and R1432H) failed to express any measurable sodium current. However, expression of a conserved positively charged mutant R1432K or a reverse-mutant G1432R (Figure 2D) resulted in a sodium current comparable to that found in WT SCN5A. Extracellular pH variations did not induce any current by the R1432H mutant, suggesting that the histidine residue may not be accessible from the extracellular side and would therefore not be affected by the extracellular pH changes. Consequently, we concluded that the hH1 mutant channel is not present at the cell surface and may be blocked within the cell. We found that the R1432G subunit transfected with or without the ß₁ subunit (data not shown) did not produce currents. Moreover, when expressed alone, the FLAG-tagged subunit of hH1 is localized to the cell surface, indicating that the -ß₁ interaction is not the cause of loss of expression. When the c-myc–tagged ß₁ subunit is expressed alone, it is retained in the perinuclear region (Figure 6B). Thus, we conclude that the subunit is required for transport of the regulatory ß₁ subunit to the cell membrane.

We report that when the R1432G mutant was expressed in oocytes, it produced a sodium current with normal gating properties (Figure 5B). Determining the importance of the positive charge at residue 1432 of hH1 channel in recognition of a chaperon trafficking protein would be crucial. This is especially true because little is known about sodium-channel trafficking. Numerous posttranslational modifications, such as the glycosylation of immature protein¹³ and the covalent attachment of fatty acid¹⁴ in sodium channels, have been previously described. Previous studies have also shown divergence in the posttranslational processing of the voltage-gated sodium channel between Xenopus oocytes and native tissue.^{14 15} Dissimilarity in functional expression between Xenopus oocytes and mammalian cells was also reported with aquaporin water channel.¹⁶ On the basis of these observations, we suggest that the localization of the newly synthesized protein could vary depending on the expression system, thus providing an explanation for the apparent abolition of functional expression of the hH1 mutant (R1432G) in the tsA201 cells. Mutations in several membrane proteins have been reported to cause defective trafficking, including ionic channels.^{17 18} Defective protein trafficking has been identified in several inheritable human diseases. Previous studies have also shown that the mutant chloride channel (F508) in patients with cystic fibrosis is retained within the cell and fails to reach the plasma membrane.^{19 20} It was recently demonstrated that 4-phenyl butyrate rescues the mutant CFTR channel trafficking defect by regulating the Hsc70 heat shock protein.²¹ In our study, the sodium currents could not be restored in the presence of 4-phenyl butyrate. This suggested that Hsc70 is not implicated in the retention of the mutated sodium channel protein inside the ER.

Several other congenital human diseases have also been shown to be caused by protein-processing defects, such as HERG potassium channel in long-QT syndrome (LQT2),^{17 22} MinK in LQT5,²³ the sodium/glucose symporter in glucose-galactose malabsorption,²⁴ lipoprotein receptors in familiar hypercholesterolemia,²⁵ and aquaporin-2 in nephrogenic diabetes.²⁶ In a recent study,²⁷ the processing of an LQTS mutant of the cardiac potassium channel (HERG) has been reported to be temperature-sensitive. In their report, Ficker et al²⁷ found that the channel trafficking defect was rescued by a lower incubation temperature (26°C instead of 37°C). In our study, temperature dependence of the protein processing was also tested. Transfected cells were incubated at 37°C, 29°C (data not shown), and 22°C for 48 hours after transfection. The absence of sodium currents in all cases ruled out the role of temperature-sensitive heat-shock chaperon protein in the processing of our mutant sodium channels.

Immunostaining and laser confocal microscopy of cells transfected with tagged channel subunits showed that the mutant protein (R1432G) was retained inside the cellular cytoplasm both in the presence of the ß₁ subunit and in its absence (Figures 6D and 6E). Our data show that the R1432G mutant is colocalized with the calnexin in the ER (Figure 7). Calnexin is a chaperon protein present in the ER vesicles.²⁸ The mechanism of the retention of mutant sodium channels hH1/R1432G is not understood. It is possible that this mutant results in a misfolded protein that experiences impaired transport from the ER to the Golgi apparatus. This localization pattern was also observed when cardiac myocytes isolated from newborn rats were transiently transfected with hH1/R1432G-FLAG mutant but not in hH1/WT-FLAG–transfected myocytes (data not shown).

In conclusion, our results suggest that the retention of the mutant channel within the ER is attributable to the absence of positive charge at position 1432. We propose that the disruption of hH1 cell-surface localization in the R1432G mutant is one mechanism that can account for the loss of functional sodium channels in Brugada syndrome.

Acknowledgments

This study was supported by the Heart and Stroke Foundation of Québec, the Canadian Institutes of Health Research MT-13181, and Fonds de la Recherche en Santé du Québec. M.C. is an Edwards Senior investigator (Joseph C. Edwards Foundation). We thank Armin Akhavan for his comments on the manuscript.

Footnotes

Original received April 24, 2001; revision received May 18, 2001; accepted May 18, 2001.

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