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Brugada Syndrome (BrS), characterized by ST segment elevation in the right precordial ECG leads and the development of life-threatening ventricular arrhythmias, has been associated with mutations in six different genes. Here, we identify and characterize a mutation in a new gene.
A 64-year-old Caucasian male displayed a Type-1 ST segment elevation in V1 and V2 during procainamide challenge. Polymerase chain reaction (PCR)-based direct sequencing was performed using a candidate gene approach. A missense mutation (L10P) was detected in exon 1 of SCN3B, the β3 subunit of the cardiac sodium channel, but not in any other gene known to be associated with BrS or in 296 controls. Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using whole-cell patch-clamp techniques. Co-expression of SCN5A/WT+SCN1B/WT+SCN3B/L10P resulted in an 82.6% decrease in peak sodium current density, accelerated inactivation, slowed reactivation and a -9.6 mV shift of half-inactivation voltage compared to SCN5A/WT+SCN1B/WT+SCN3B/WT. Confocal microscopy revealed that SCN5A/WT channels tagged with green fluorescent protein (GFP) are localized to the cell surface when co-expressed with WT SCN1B and SCN3B, but remain trapped in intracellular organelles when co-expressed with SCN1B/WT and SCN3B/L10P. Western blot analysis confirmed the presence of NaVβ3 in human ventricular myocardium.
Our results provide support for the hypothesis that mutations in SCN3B can lead to loss of transport and functional expression of the hNav1.5 protein, leading to reduction in sodium channel current and clinical manifestation of a Brugada phenotype.
Brugada syndrome (BrS) is a cardiac channelopathy characterized by ST segment elevation or the appearance of accentuated J waves in the right precordial leads (V1–V3) of the ECG and the development of life-threatening polymorphic ventricular tachycardia (VT). The electrocardiogram characteristics of the BrS are dynamic and often concealed but can be unmasked by potent sodium channel blockers1,2.
BrS has been associated with mutations in six different genes. Mutations in SCN5A (Nav1.5, BrS1) have been reported in 14.3-15% of BrS probands, CACNA1C (Cav1.2, BrS3) in 6.7%, CACNB2b (Cavβ2b, BrS4) in 4.8% and mutations in Glycerol-3-phophate dehydrogenase 1-like enzyme gene (GPD1L, BrS2), SCN1B (β1-subunit of sodium channel, BrS5) and KCNE3 (MiRP2; BrS6) are much more rare3-8. These genetic defects lead to development of BrS secondary to either a loss of function of sodium (INa) or L-type calcium (ICa) channel current, or a gain of function of transient outward current (Ito). Thus, approximately 72% of BrS probands remain genotype-negative. Here, we report the identification of another gene associated with the BrS phenotype caused by loss-of-function of INa secondary to a mutation in the β3-subunit of the cardiac sodium channel, encoded by SCN3B.
Genomic DNA was prepared from peripheral blood lymphocytes of the patient. Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit (Gentra System, Puregene, Valencia, CA, USA). All known exons of the Brugada-susceptibility genes (SCN5A, IRX5, SCN1B, SCN3B, CaCNB2B, CACNA1C, KCNE2, KCNE3, GPD1L) were amplified with intronic primers and sequenced in both directions to probe for mutations, with the use of an ABI PRISM 3100-Avant Automatic DNA sequencer (Applied Biosystem. Foster City, CA). The sequence primers of SCN3B are shown in Table 1 (Reference Sequence: NM_018400). One hundred twenty individuals, matched by race and ethnic background, with no history of cardiac arrhythmias were used as controls.
For patch-clamp study, site-directed mutagenesis was performed with QuikChange (Stratagene, La Jolla, CA) on full-length human wild-type (WT) and mutant SCN3B cDNA cloned in pIRES2-DsRed-Express (RFP) vector, the WT SCN1B cloned in pIRES2-AcGFP1 vector, and the WT SCN5A cloned in pcDNA3.1. SCN3B was a kind gift from Dr. Takashi Tokino, Japan. In the case of trafficking studies, human WT SCN5A cDNA cloned in pcDNA3.1 with fusion green fluorescent protein (GFP) at the C-terminus was co-expressed with, the WT SCN1B cloned in pRC-CMV, and WT and mutated SCN3B cloned in pIRES2 with RFP. The mutated plasmid was sequenced to ensure the presence of the mutation without spurious substitutions.
Sodium channels were expressed in a modified human embryonic kidney cell line, TSA201. Briefly, transient transfection using fugene6 (Roche Diagnostics, Indianapolis, IN). was carried out with SCN5A, SCN1B and SCN3B (WT or mutant) with a molar ratio of 1:1:1. The cells were grown in GIBCO DMEM medium (No. 10566, Gibco, Invitrogen cell culture, Carlsbad, CA) with FBS (No. 16000) and antibiotics (No. 15140) on polylysine coated 35 mm culture dishes (Cell+, Sarstedt, Newton, NC, USA). Cells were placed in a 5% CO2 incubator at 37°C for 24 to 48 hours prior to patch clamp study. It is noteworthy that previous studies have demonstrated that no endogenous SCN5A and its beta-subunits are expressed in the TSA201 cell line9.
Membrane currents were measured using whole-cell patch-clamp techniques. All recordings were obtained at room temperature (20 - 22°C) using an Axopatch 200B amplifier equipped with a CV-201A head stage (Axon Instruments Inc., Foster City, CA). Measurements were started 10 minutes after obtaining the whole-cell configuration to allow the current to stabilize. The holding potential was maintained at -120 mV. Macroscopic whole cell Na+ current was recorded by using bath solution perfusion containing (in mmol/L) 130 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 2.8 Na Acetate, 10 HEPES, 10 Glucose (pH 7.3 with NaOH). Tetraethylammonium Chloride (5 mmol/L) was added to the buffer to block TEA-sensitive native currents. Patch pipettes were fabricated from 1.5 mm OD borosilicate glass capillaries (Fisher Scientific, Pittsburgh, PA). They were pulled using a gravity puller (Model PP-830, Narishige Corp, Japan) to obtain resistances between 0.8 - 2.8 MΩ when filled with a solution containing (in mmol/L) 5 NaCl, 5 KCl, 130 CsF, 1.0 MgCl2, 5 EGTA, 10 HEPES and 5 TEA (pH 7.2 with CsOH). Currents were filtered with a four pole Bessel filter at 5 kHz and digitized at 50 kHz. Series resistance was electronically compensated at around 80%.
INa was elicited by depolarizing pulses ranging from -90 mV to +30 mV in 5 mV increments with a holding potential of -120 mV. Peak currents were measured and INa densities (pA/pF) were obtained by dividing the peak INa by the cell capacitance obtained. Activation properties were determined from I/V relationships by normalizing peak INa to driving force and maximal INa, and plotting normalized conductance vs. Vm. Voltage-dependence of steady-state inactivation was obtained by plotting the normalized peak current (40-ms test pulse to −20 mV after a 1000-ms conditioning pulse from -140 mV to -60 mV with the holding potential of -120 mV) vs. Vm. The activation and steady-state inactivation curves were fitted to the Boltzmann equation, I/Imax= 1/(1+exp((V-V1/2)/k)) to determine the membrane potential for half-maximal inactivation V1/2 and the slope factor k. Pulses for recovery from inactivation were of 20 ms duration. Peak current elicited during the second pulse was normalized to the value obtained during the initial test pulse. It was analyzed by fitting data to a double exponential function: I(t)/Imax=Af(1-exp(-t/τf))+As(1-exp(-t/τs)), where Af and As are the fractions of fast and slow inactivating components, respectively, and τf and τs are their time constants.
All data acquisition and analysis were performed using pCLAMP V9.2 (Axon Instruments, Foster City, CA), EXCEL (Microsoft) and ORIGIN 7.5 (Microcal Software, Northampton, MA, USA).
We assessed channel trafficking using Na+ channels α-subunit (SCN5A) tagged with GFP. Confocal microscopy was used to localize the channels and identify trafficking defects. Briefly, cells were grown on polylysine coated glass bottom 35mm culture dishes and studied 48 hours post-transfection. Experiments were performed on an Olympus FluoView laser-scanning confocal microscope (Olympus, Orangeburg, NY, USA) and images were acquired with Fluoview acquisition software program on a personal computer. GFP-labeled cells were analyzed in the XYZ configuration. An argon laser provided the excitation light at 488 nm and the emission light was collected at 520nm in photomultiplier tube (PMT) #1. Transmission image was acquired in PMT #2. Fluorescence signals were collected with either a 40× or a 60× oil-immersion objective lens. XY frame was set to 512 × 512 pixels and laser intensity was set to 6% power. The Z-axis was changed in approximately 0.50 μm increments by computer control through the entire volume of the cell. To quantify the membrane expression of Nav1.5 fluorescence intensity at the plasma membrane region (2 μm) and the entire cell area in the middle XY image of the Z series stack was measured and the ratio of peripheral to total cell area fluorescence was calculated. Analysis of GFP-labeled cells was performed using both Fluoview and Image J software.
Membrane proteins (50 μg/lane, except for TSA201-SCN3B 0.5 μg/lane) were run on 5 – 15% gradient linear SDS-PAGE. BIO-RAD 161-0374 Precision Plus Protein Dual Color Standards 10 λ/lane was used as molecular marker reference. Rabbit Anti-Human SCN3B Polyclonal Antibody at 1:500 (ab48552, Abcam, Cambridge, MA) was used to detect bands in membrane proteins from untransfected TSA201 cells (negative control), SCN3B transfected TSA201 cells (positive control) and from two human left ventricular samples.
Data are presented as Mean ± SEM. A two-tailed Student's t-test was used for statistical comparison of two groups and ANOVA coupled with Student-Newman-Keuls test for comparison of three or more groups (SigmaStat, Jandel Scientific Software). Differences were considered statistically significant at a value of P<0.05.
The study was approved by the Regional Institutional Review Board. The proband, a 64 year old Caucasian male (German, Swedish and Native American descent), presented with a resting ECG displaying a slight ST segment elevation and negative T wave in V1 suggestive, but not diagnostic, of BrS. A Type 1 ST-segment elevation, diagnostic of a BrS, was unmasked in leads V1 and V2 with sodium channel blockade using procainamide (Figure 1). ECG characteristics are summarized in Table 2. PR interval was 180 ms at baseline and increased to 200 ms after procainamide. An internal cardioverter-defibrillator (ICD) was implanted in 2005. The proband was asymptomatic and did not have a family history of sudden cardiac death (SCD). Family history was remarkable for a high incidence of cancer. The proband had 2 paternal aunts and 3 paternal uncles, all of whom died of lung cancer. Interrogation of the ICD in July of 2008 revealed an episode of atrial flutter with 2:1 AV block.
Genetic analysis of 9 Brugada-susceptibility genes (SCN5A, SCN1B, CaCNB2B, CaV1.2, IRX5, KCNE2, KCNE3, KCNE4, GPD1L) proved negative. Further genetic screening of the sodium channel β-subunits revealed a novel missense mutation in exon 1 of SCN3B. Polymerase chain reaction (PCR)-based sequencing analysis revealed a double peak in the sequence of exon 1 of the SCN3B gene (Figure 2A) showing a T-to-C transversion at nucleotide 28, predicting a leucine (L) to proline (P) substitution at residue 10 (designated L10P). This nucleotide change was not observed in 360 Caucasian, 120 Turkish and 112 Sephardic Jews reference alleles, suggesting that this variation is rare in the general population. Leu-10 is located in the extra cellular domain (ECD) of SCN3B (Figure 2B) and is highly conserved through evolution (Figure 2C). The only family member available for study was an unaffected brother whose ECG was normal except for right bundle branch block (RBBB). The brother was negative for the L10P SCN3B mutation. The proband was the only case in our series of 179 BrS probands with a mutation in SCN3B. 129 of the 179 (72.07%) probands tested negative for all genes tested.
SCN5A/WT+SCN1B/WT, SCN5A/WT+SCN1B/WT+SCN3B/WT, or SCN5A/WT+SCN1B/WT+SCN3B/L10P were expressed in TSA201 cells to assess the effects of the mutation on sodium channel function. Figure 3A shows macroscopic currents recorded from these channels together with the current-voltage (I-V) relationships. Maximum peak inward current occurred at a potential of -35 mV for all channel types. Co-expression of SCN3B/WT with SCN5A/WT+SCN1B/WT increased peak current density from -281.3±62.3 pA/pF to -402.8±93.2 pA/pF (n=9 and 13, respectively; P <0.05 between 2 groups). Co-expression of SCN3B/L10P resulted in a marked decrease in peak sodium current density to -70.2±14.5 pA/pF (n=25; 17.4% of SCN5A/WT+SCN1B/WT+SCN3B/WT and 25.0% of SCN5A/WT+SCN1B/WT current density; P< 0.05 for each; Figure 3B). Co-expression of SCN3B/L10P produced total loss of function in 40 % of cells studied (10 out of 25 cells, Figure 3A).
The half-inactivation voltage (V1/2) of mutant INa channels (SCN5A/WT+SCN1B/WT+SCN3B/L10P) was 14.8 and 9.6 mV more negative than those of SCN5A/WT+SCN1B/WT and SCN5A/WT+SCN1B/WT+SCN3B/WT channels, respectively. (P<0.01 respectively; see in Table 3 and Figure 4B). Steady-state activation, obtained after applying the step protocol in inset of Figure 3A, was similar among the 3 groups (Figure 4B). Recovery from inactivation, measured using a standard double paired-pulse protocol, was similar in the two control groups but slower in the mutant channels (P<0.01 respectively; see in Table 3 and Figure 4C). The L10P mutation caused a shift in the voltage –dependence of steady-state inactivation and slowed recovery from inactivation, thus serving to further reduce sodium channel availability.
To evaluate whether the loss of function caused by the SCN3B/L10P mutation is due in part to a trafficking defect, we studied GFP-fusion-SCN5A/WT co-expressed with SCN1B/WT alone, or combined with either SCN3B/WT or SCN3B/L10P. XYZ scans of SCN5A/WT+SCN1B/WT on the confocal microscope revealed both a central and peripheral pattern of staining suggesting localization of the channel in the cell membrane as well as intracellular organelles (Figure 5A-C). Protein expression of SCN5A/WT channels was enhanced when both SCN1B/WT and SCN3B/WT were added (Figure 5D-F). In contrast, SCN3B/L10P resulted in internal staining consistent with intracellular compartmentalization with no evidence of plasma-member staining (Figure 5G-I), suggesting that channels were trapped in the endoplasmic reticulum and/or Golgi complex. The ratio of peripheral to total cell area fluorescence intensity was similar for SCN5A/WT+SCN1B/WT and SCN5A/WT+SCN1B/WT+ SCN3B/WT (P>0.05), but significantly reduced for the mutant channel (SCN5A/WT+SCN1B/WT+ SCN3B/L10P; P<0.01; Figure 5J). These results demonstrate that β3-subunit mutations can lead to impaired trafficking of the cardiac sodium channel.
Figure 6 shows the results of Western blot analysis performed to confirm the presence of Naβ3 in human ventricular myocardium. Anti-Human SCN3B Antibody detected a unique band in membrane proteins from SCN3B transfected TSA201 cells (positive control) and from two human left ventricular myocardial preparations, but not in untransfected TSA201 cells (negative control).
Voltage-gated sodium channels are vital to the function of excitable cells including those comprising the heart. Auxiliary or β subunits are known to provide functional diversity among sodium channels. They do not form the ion-conducting pore, but are multifunctional proteins that play critical roles in modulation of channel function, regulation of channel expression levels at the plasma membrane, as well as cell adhesion10. To date, four different Navβ subunits have been described (SCN1B, SCN2B, SCN3B, and SCN4B)11-15, and shown to play a critical role in cell adhesion, signal transduction, channel expression at the plasma membrane, and voltage dependence of channel gating16-19. All are detectable in cardiac tissue9. β1A, a splice variant of SCN1B, is expressed in embryonic brain and adult heart in rat20. The distribution and expression level of sodium channel α and β subunits in human and canine hearts has not been not well characterized.
Because of their significant role in modulating channel expression and function, genes that encode cardiac channel β subunit proteins are attractive candidates for ion channelopathies like BrS21. The role of β1 subunits have been studied most extensively. β1 co-expression has been reported to have no observable effect on SCN5A function22,23, to result in increased sodium current density with no detectable effects on channel kinetics or voltage-dependence24,25, to modulate channel sensitivity to lidocaine block with subtle changes in channel kinetics and gating properties26 as well as to shift the voltage-dependence of steady-state inactivation27-29 or alter the rate of recovery from inactivation29,30.
Previous studies involving co-expression of SCN5A with β3 have reported 1) increased current density, a depolarizing shift in the voltage-dependence of inactivation, and an increased rate of recovery from inactivation in Xenopus oocytes30, or 2) a hyperpolarizing shift of inactivation, slowed recovery from inactivation, and reduced late sodium channel current, without any change in peak current density in Chinese hamster ovary (CHO)-K1 cells29. The present study involving TSA201 cells, shows that co-expression of β3 with SCN5A and β1 increases peak sodium current density, shifted the voltage dependence of channel availability in a hyperpolarizing direction without significantly changing the voltage-dependence of activation or recovery kinetics (Figs. 3 and and4,4, Table 3).
Although mutations in Nav1 α subunits have been associated with inherited diseases, including LQT3, Brugada syndrome, progressive conduction disease and atrial standstill31, to date only 2 genes encoding sodium channel β-subunits have been associated with human cardiac disease7,32.
It has long been appreciated that SCN5A mutations associated with LQTS and Brugada syndrome are modulated by co-expression of SCN1B33. Brugada syndrome is known to be caused by a reduction in INa. The role of β1 subunits to exacerbate loss of function produced by R1232W/T1620M mutations in SCN5A in patients with BrS was demonstrated by Wan et al34.
Mutations in β1 subunits (Navβ1 and Navβ1b) have recently been shown to be associated with a combined Brugada syndrome and cardiac conduction disease phenotype in humans7. Mutation in β4 subunit have been reported to be associated with an increase in late INa giving rise to the LQT3 variant of the long QT syndrome.32
In the present study, we provide evidence that SCN3B is a BrS-susceptibility gene. An L10P missense mutation in a highly conserved residue, absent in 240 reference alleles, is shown to produce a major reduction in INa secondary to both functional and trafficking defects in cardiac sodium channel expression. It is well established that there are 4 fully conserved cysteine residues, labeled C2, C21, C24, and C96 in β1 and β3 subunit15. The disulfide bonds between C2 and C24, C21 and C96 are believed to correspond to the interaction site for α-subunit association. Moreover, the former bond is also responsible for forming the Ig fold for β1 and β3, a disruption of which can cause an inherited epilepsy syndrome. Our mutation (L10P) is located near C2, which may affect the interaction of the β3 subunit with the sodium channel complex, and lead to the phenomenon that we observed. Little is known about cardiac sodium channel α- and β-subunit trafficking in vivo. A study using over-expression of fluorescent-tagged SCN5A, β1, and β2 in HEK293 cells suggested that SCN5A and β2 are transported separately to the plasma membrane while SCN5A and β1 form a complex in the endoplasmic reticulum (ER) that may facilitate plasma membrane trafficking35. In PC12 and CHO-K1/Nav1.5 cells, enhanced green fluorescent protein (EGFP)-tagged ΔECD (extracellular domain-deletion) β3 mutant showed internal staining with little plasma membrane staining, and EGFP-tagged ΔICD (intracellular domain deletion) β3 mutant showed no evidence of surface staining but labeled an internal highly reticulated compartment that suggests ER. These results indicate that a mutation in the extracellular domain can impair trafficking of SCN3B to the membrane and that deletion of the intracellular domain totally disrupts trafficking of the β subunit. Our results suggest that WT β3 plays a role in facilitating SCN5A transport to the plasma membrane, since a mutation in the extracellular domain of β3 is capable of disrupting trafficking of SCN5A to the plasma - membrane.
An interesting aspect of the family studied was that many family members died of lung cancer. SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of tumor suppressor p53, a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation, and adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. These results suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs36. A more recent study discovered that SCN3B is a candidate cancer gene which could affect ion channel transport37. In view of the above, it is tempting to speculate that the L10P mutation in SCN3B may also promote cancer and thus contribute to death of mutant carriers in this family. This hypothesis remains to be tested.
A genotype-phenotype correlation between the β3 mutation and BrS phenotype is hampered by the high incidence of cancer deaths in this family, which may be caused by the same mutation in the β3 subunit. Only two family members were available for study. A native American cohort was not available for study as as a control group.
Another limitation of the study is the fact that characterization of the SCN3B mutation were carried out in a heterologous mammalian expression system, creating conditions which may be different from those encountered in vivo as far as a contribution of β2 or β4 subunits, or other components of the Na channel macromolecular complex. Despite these limitations, the electrophysiological characteristics of the mutant channel are concordant with the BrS phenotype, and in combination with the clinical data supports a causal relationship between the L10P mutation in SCN3B and the disease.
Our results provide evidence in support of the hypothesis that mutations in the SCN3B-encoded Navβ3 subunit constitute another pathogenic mechanism responsible for development of the Brugada syndrome phenotype secondary to a loss of function of cardiac sodium channel current.
The authors are grateful to Judy Hefferon and Robert J. Goodrow, Jr. for technical assistance and to Susan Bartkowiak for maintaining our Genetic Database.
FUNDING SOURCES: Supported by a grant # HL 47678 (CA) from the National Institutes of Health and the Grand Lodges of New York State and Florida.
Conflict of Interest Disclosures None.
The Brugada Syndrome (BrS) is an inherited cardiac arrhythmia syndrome associated with a high incidence of sudden cardiac arrest. This disorder has previously been linked to mutations in six different genes: SCN5A, GPD1L, CACNA1c, CACNB2b, SCN1B and KCNE3. The present study provides evidence that a mutation in SCN3B, encoding the β3 subunit of the sodium channel, can cause a loss of function in INa leading to a Brugada phenotype. The genes thus far associated with Brugada syndrome lead to either a loss of function in sodium or calcium channel current (INa and ICa) or to a gain of function in transient outward current potassium current (Ito). The decrease in inward current or increase in outward current cause a shift in the balance of current flowing during the early phases of the cardiac action potential (AP) leading to accentuation of the AP notch in the epicardium but not in the endocardium. The resultant transmural gradient leads to an accentuation of the electrocardiographic J wave, manifest also as an ST segment elevation, and the development of phase 2 reentry and polymorphic ventricular tachycardia. This Brugada syndrome phenotype is most commonly limited to the right precordial leads because Ito is usually most prominent in the right ventricular outflow tract. Understanding the genetic basis for the Brugada syndrome may assist with the diagnosis and ultimately with the approach to therapy.