A Novel Missense Mutation, I890T, in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na+ channel alpha subunit (Nav1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Nav1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Nav1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from −52.0±6.5 pA/pF, n = 15 to −35.9±3.4 pA/pF, n = 22, at −20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V 1/2 = −32.0±0.3 mV, n = 18, and −27.3±0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.


Introduction
Alterations of the sodium current (I Na ) in the human heart can lead to diseases responsible for cardiac arrhythmias, such as Brugada Syndrome (BrS) [1]. This syndrome, first described in 1992, is characterized by the presence of ST segment elevation in the right precordial leads (V1-V3) of the electrocardiogram (ECG), without major structural alterations in the heart [2]. The prevalence of BrS is in the range of 1-5 in every 10,000 individuals and is an important cause of Sudden Cardiac Death (SCD) [3].
Since the discovery of the first genetic variation in the cardiac sodium channel gene, SCN5A, associated with BrS [4], many studies have classified this syndrome as a genetic disease with autosomal dominant inheritance and incomplete penetrance [5]. It has been demonstrated that mutations in SCN5A associated with BrS result in loss-of-function of the current carried by the cardiac type sodium channel (Na v 1.5) [6]. Different mechanisms are known to produce channel loss-of-function, including reduced expression of the channel in the plasma membrane, changes in the voltage dependence of the channel activation or inactivation, or altered channel kinetics [7]. In addition, mutations in genes other than SCN5A have been identified in a low proportion of BrS patients [8].
The Na v 1.5 protein, with 2016 amino acids and a molecular weight of 227 kDa, consists of four homologous domains (DI-DIV) [9]. Each domain contains six transmembrane segments (S1-S6) linked by intracellular and extracellular loops. S4 segments contain 5 positively charged residues (arginine or lysine) separated by 2 hydrophobic residues, and form the voltage sensor domain of the channel. The pore region of the channel is formed by the interaction among segments S5, S6 and loop S5-S6 of domains DI to DIV [10]. The pore (P)-helices that stabilize the Na + ion in the central cavity are formed by the loops S5-S6 [11].
In the present study, we aimed to characterize the biophysical properties of Na v 1.5 channels carrying a novel mutation, I890T, in the first P-helix of DII to establish whether this mutation is associated with BrS. We show evidence of loss-of-function of the mutant Na v 1.5 channel, which is consistent with the patient's clinical manifestation of BrS.

Ethics Statement
This study was approved by the Ethics Committee of Hospital Josep Trueta (Girona, Spain) and conforms with the principles outlined in the Declaration of Helsinki. All individuals signed a written informed consent to participate in the study.

Reagents
All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise.

Genetic Analysis of SCN5A
Total genomic DNA was isolated from blood samples using the Puregene DNA purification Kit (Gentra Systems, Minneapolis, MI, USA). The exons and exon-intron boundaries of SCN5A were amplified (Verities PCR, Applied Biosystems, Austin, TX, USA), the PCR products were purified (Exosap-IT, USB, Isogen Life Science, The Netherlands) and they were directly sequenced in both directions (Big Dye Terminator v3.1 cycle sequencing Kit and 3130XL Genetic Analyzer, both from Applied Biosystems). The DNA sequence was compared with the reference sequence NM000335 for SCN5A (OMIM601144) (UCSC Genome binformatics [12]/NCBI-Mendelian Inheritance [13]). DNA samples from 300 healthy Spanish individuals (600 alleles) were used as control samples.
The resultant construct was directly sequenced to verify the presence of the desired mutation and the absence of additional variations.
pcDNA3.1 included a FLAG tag (sequence: DYKDDDDK between prolines P154 and P155 of SCN5A) which has been previously shown not to alter the Na v 1.5 current properties [14,15]).
HEK cells were transiently transfected with 2.9 mg of the SCN5A construct, either WT or I890T, using GeneCellin TM Transfection Reagent (BioCellChallenge, Toulon Cedex, France) following the manufacturer's specifications. Co-transfection with 0.1 mg of a plasmid containing the green fluorescent protein (GFP) gene (a kind gift from Dr. Kirstine Callø, University of Copenhagen, Copenhagen, Denmark) allowed the identification of transfected cells. All experiments were performed 48 hours after transfection.
Activation curve data were fitted to a Boltzmann equation, of the form g = g max /(1+ exp(V 1/2 2V m )/k), where g is the conductance, g max the maximum conductance, V m is the membrane potential, V 1/2 is the voltage at which half of the channels are activated and k is the slope factor. Steady-state inactivation values were fitted to a Boltzmann equation of the form I = I max /(1+ exp(V 1/2 2V m )/k), where I is the peak current amplitude, I max the maximum peak current amplitude, V m is the membrane potential, V 1/2 is the voltage at which half of the channels are inactivated, and k is the slope factor. The sodium current decay after the peak I Na was fitted with a monoexponential function between 240 and 225 mV, and a bi-exponential function between 220 and 20 mV, from where t fast and t slow were obtained. Both the slow inactivation and the recovery from inactivation data were fitted to mono-exponential functions, to obtain their respective time constants.

Cell Surface Protein Biotinylation
Cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS) supplemented with 0.9 mM CaCl 2 and 0.49 mM MgCl 2 (DPBS + ) at pH 7.4. Membrane proteins were biotinylated by incubating cells with 1.6-2.5 mg/ml of EZ-link sulfo-NHS-LC-LC-biotin (Pierce, Thermo Scientific, Rockford, IL, USA) in DPBS + for 30 min at 4uC. Cells were then washed 3 times in DPBS + with 100 mM glycine, then with DPBS + containing 20 mM glycine, and scrapped in Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and Complete Protease Inhibitor Cocktail (Roche, Madrid, Spain)). Lysates were obtained after 1 h rotating at 4uC. Insoluble materials were removed by centrifugation. Supernatants were incubated with Ultralink Immobilised NeutrAvidin beads (Pierce) overnight at 4uC. The beads were precipitated and washed with Triton X-100 lysis buffer, then in saline solution (5 mM EDTA, 350 mM NaCl and 0.1% TX-100 in DPBS + pH 7.4) and finally in 10 mM Tris/HCl pH 7.4. Precipitated beads were resuspended in SDS-PAGE loading buffer and heated for 5 min at 70uC. Proteins were resolved in 4% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were probed with a rabbit anti-human Na v 1.5 antibody (anti-hNa v 1.5; Alomone Labs, Jerusalem, Israel) at a dilution of 1:1,000, overnight at 4uC. A secondary horseradish peroxidase-conjugated antibody (Thermo Scientific, Rockford, IL, USA) was used at a dilution of 1:2,000 for 1 h at room temperature, and signals were detected with the SuperSignal West Femto Chemiluminiscent substrate (Pierce). A mouse antibody against Na + /K + ATPase was used as biotinylation control. Protein markers for molecular weights from 10 to 250 kDa (PageRuler TM Plus Prestained Protein Ladder, Thermo Scientific, Rockford, IL, USA) were used as size standards in protein electrophoresis (SDS-PAGE) and Western blotting.
Expression of Na v 1.5 was quantified using the ImageJ software (National Institute of Health, NIH) available at http://rsb.info. nih.gov/ij. Intensity values for each band were determined as the integrated density (sum of pixel values) within a fixed area. To account for differences of these values between WT and I890T due to loading, I890T intensity values were normalized with the ratio between WT and I890T Na + /K + ATPases.

In silico Studies of I890T
The software tools ESyPred3D 1.0 [18], Modeller 9.9 [19] and CPHmodels [20] were used to build a model of the pore module of DII of Na v 1.5, based on the structure of the bacterial voltage-gated sodium channel (Na v Ab) ( [11]). The model was constructed as a chimera of Na v Ab and Na v 1.5 as follows: the sequence of S1 to S4, as well as the loop S4-S5, was that of Na v Ab; the sequence of S5, loop S5-S6, and S6 was that of DII of Na v 1.5. No further constraints were defined.

Statistical Analyses
Results are presented as means 6 standard error (SE). Statistical comparisons were performed using the unpaired Student's t-test. Results are considered statistically significant when p,0.05.

Identification of I890T, a Novel Na v 1.5 Channel Mutation
The proband, a 31-year-old Spanish male, was admitted to the hospital due to the suspicion of BrS during a routine examination. His baseline 12-lead ECG showed a ST segment elevation in leads V1-V3 that strongly suggested BrS type I (Fig. 1A). He had suffered an episode of syncope at the age of 12.
A heterozygous variation (thymine-to-cytosine) at position 2669 of the SCN5A gene (c.2669 T.C) was identified in the proband's DNA (Fig. 1B). This base transition leads to an isoleucine-tothreonine substitution at position 890 (p.I890T) of the Na v 1.5 channel. This genetic variation was absent in 600 control alleles of the same ethnic background, and was not found in the Human Gene Mutation Database (HGMD) [21], Ensembl [22], HapMap [23], 1000 genomes project [24] and NHLBI Exome Sequencing Project [25]. The I890T variation in Na v 1.5 channel thus represents a possible novel mutation causing BrS.
The genetic study of the proband's family members revealed that two sisters had the I890T mutation (Fig. 1A). A 34-year-old sister presented several episodes of syncope, her baseline ECG was normal, but a ST segment elevation in lead V2 characteristic of BrS was unmasked upon ajmaline challenge, thus confirming BrS. A 18-year-old sister was asymptomatic, had a normal baseline ECG, and presented no alterations after challenge with either flecainide or ajmaline. The proband's third brother refused to undergo genetic analysis despite having a previous history of syncopes. The proband's mother carried the I890T mutation. She presented an episode of syncope at age 18, but showed a normal ECG when subjected to drug challenge tests. The proband's father did not carry the mutation and was asymptomatic, and had a normal ECG after drug provocation test.
To further explore the different clinical phenotypes found among the carriers of the I890T mutation, we looked for other genetic variations in the SCN5A gene in the family. We found that the younger sister, who did not present any cardiac abnormalities, carried a non-synonymous polymorphism, p.H558R, inherited from the father. This variation was not found in any of the other mutation carriers.

I890T Markedly Decreases Peak I Na and Modifies Na v 1.5 Channel Activation Kinetics
We conducted patch-clamp studies to assess the effect of the mutation I890T on whole cell sodium currents. HEK cells were transfected with either WT or I890T channel constructs (referred to as WT cells and I890T cells, respectively, in the remaining text). Current traces in Figure 2A show that I Na is substantially reduced by the mutation I890T. This reduction was confirmed by analysis of peak I Na density, which showed a significant decrease in the current-voltage relationship (I-V) of I890T cells with respect to WT cells (Fig 2B, Table 1).
In addition, we observed a positive shift of the activation curve towards more positive potentials (Fig. 3), which further contributes to the loss-of-function of I890T channels. Data fitting to a Boltzmann equation revealed a significant 5 mV shift of V 1/2 in I890T respect to WT cells, whereas no changes were observed in the slope factor (Table 1).
Next, we assessed the voltage-dependence of steady-state inactivation for WT and I890T cells. The mutation I890T did not affect the voltage dependence of channel availability (Fig. 3, Table 1).
Analysis of the time courses of inactivation, slow inactivation and recovery from inactivation are illustrated in Figure 4. Inactivation time constants were obtained from fitting the time Activation and steady-state inactivation parameters were calculated by data fitting to Boltzmann functions (see Methods). V 1/2 is the voltage for half-maximal activation or steady-state inactivation and k is the slope factor. Slow inactivation and recovery from inactivation data were fitted to mono-exponential functions (see Methods) to obtain the time constant t. Values are expressed mean 6 SE. *p,0.05; **p,0.01. doi:10.1371/journal.pone.0053220.t001 course of the currents elicited with the stimulation protocol used for the I-V relationship, and plotted as a function of voltage (Fig. 4A). The time constants for I890T and WT currents remained similar at the voltage range analyzed. Double pulse protocols were used to study I Na slow inactivation and recovery from inactivation. No differences were found either in the slow inactivation or recovery from inactivation parameters obtained from fitting of data from WT and I890T cells to mono-exponential functions ( Fig. 4B and 4C, respectively, and Table 1).

I890T does not Alter Membrane Expression of Na v 1.5 Channel
I890T caused a significant decrease in I Na (Fig. 2). Thus, we tested whether this change in I Na was caused by alterations in Na v 1.5 expression in the plasma membrane. Figure 5A shows western blot bands obtained after cell surface protein biotinylation from 6 independent experiments. Densitometry analysis of the bands ( Fig. 5B and C) did not reveal statistically significant differences between WT and I890T cells (Na v 1.5 I890T/WT was 1.1160.14, n = 6).
Taken collectively, our data indicate that the I890T mutation causes a loss-of-function of Na v 1.5 current by the modification of the biophysical properties intrinsic to channel activity, rather than by impaired expression at the plasma membrane.
In silico Models of the Pore Region of WT and I890T DII of Na v 1.5 We performed a sequence alignment between DII of Na v 1.5 channel and the corresponding sequence of Na v Ab channel (Fig. 6A, upper panel) and built models for the S5-S6 region of the human channel using protein structure prediction models. Figure 6B shows the prediction obtained using the CPHmodels tool [20]. The loop S5-S6 in our model had a longer, flexible turret loop before the P1-helix. I890 resided within the second turn of the P1-helix, buried among the turret loop, the selectivity filter and the P2-helix.The structure of the S5-S6 loop was not apparently affected by the introduction of the I890T mutation (not shown).

Discussion
In the present work we identified a novel SCN5A mutation in a patient diagnosed with BrS. The same mutation was found in three other family members, two of whom presented signs of potential arrhythmogenicity. Complete genetic analysis of the SCN5A gene revealed that the younger sister, who did not present any cardiac abnormalities, carried a common polymorphism (p.H558R), inherited from the father. This variation was not found  (Fig. 2) was used to determine inactivation time constants in the voltage range between 240 and 20 mV. Current decay after the peak I Na was fitted to a monoexponential function (from 240 to 225 mV) and a bi-exponential function (from 220 to 20 mV), and the resulting time constants (t) were plotted versus the applied voltage for WT and I890T. (B) Voltage dependence of slow inactivation for WT and I890T were studied by applying the double protocol pulse shown in the inset. A 20-970 ms conditioning pre-pulse to 220 (P1) was followed by a 20 ms hyperpolarization to 2120 mV, to recover fast-inactivated channels, and then a 20 ms test pulse to 220 mV (P2). The peak current ratio P2/ P1 was plotted against the P1 prepulse duration, and data was fitted to mono-exponential functions (solid lines). (C) Recovery from inactivation properties for WT and I890T were studied by applying the double pulse protocol shown in the inset. A 50 ms depolarizing pulse to 220 mV (P1) was followed by a hyperpolarizing pulse to 2120 mV of increasing duration (1-30 ms), that preceded a test pulse to 220 mV (P2). The P2/ P1 ratio values plotted against the recovery interval times were fitted to mono-exponential functions (solid lines). A, B and C: Values are expressed as mean 6 SE. Symbols represent values for WT (filled symbols) and I890T (open symbols). doi:10.1371/journal.pone.0053220.g004 in any of the other mutation carriers. One of the most intriguing features of BrS is the marked phenotypic variability. Clinical phenotype of individuals that carry SCN5A mutations may range from asymptomatic to SCD [26]. It has been postulated that modifying factors, such as genetic background and environment, influence the clinical phenotype of BrS patients [27,28]. Several factors may explain the absence of symptoms in the younger sister. Young age and being a female are factors that diminish risk of arrhythmogenesis in BrS patients. In addition, the younger sister carries the polymorphism p.H558R which has been identified as a palliative factor in the pathological effects of BrS associated mutations [29,30]. Still, this polymorphism was not found in the mother who, despite having suffered syncope, was not diagnosed with BrS. It is evident from these data that a combination of modifying factors is determinant of the clinical phenotype, especially when the functional effect of the pathogenic mutation is mild.
The c.2669 T.C nucleotidic change produces an amino acidic variation of an isoleucine-to-threonine in position 890 (p.Iso890Thr, p.I890T), localized in the P-loop of the domain II of the Na v 1.5 channel (Fig. 6C). Sequence alignment demonstrated that I890 is conserved within the members of the voltage-gated sodium channel a-subunit family as well as in Na v 1.5 channels of Figure 5. Membrane expression of Na v 1.5 channel is not affected by I890T. Western blot detection of Na v 1.5 and Na + /K + ATPase proteins performed after cell surface biotinylation from WT and I890T cells. (A) Image shows the bands obtained for Na v 1.5 and Na + /K + ATPase, in 6 independent experiments from cells expressing either WT or I890T. Position of the markers is shown on the left side. Numbers correspond to each experiment. (B) Bar graph depicts the Na v 1.5 intensity values normalized by the Na v 1.5 WT intensity values (n = 6). Na v 1.5 I890T intensity values were previously corrected by multiplying the raw integrated density values by the ratio between the WT and I890T Na + /K + ATPase integrated density values. Note that for experiment number 5, the ratio between I890T and WT was calculated from the average of the intensity values obtained for the two samples of each condition. (C) Bar graph shows the average intensity values (expressed as mean 6 SE) obtained in (B). doi:10.1371/journal.pone.0053220.g005 other species (Fig. 6A, middle and lower panels), indicating that a hydrophobic residue is essential at this position.
A correlation between the region of the Na v 1.5 channel where mutations are located and the severity of the clinical phenotype has not been well established. Meregalli et al. [31] published a systematic study showing a correlation between the type of mutation and the changes produced in Na v 1.5 currents. A broader study would most likely give light to this intriguing subject. Unfortunately, this type of study is difficult to achieve mainly due to the fact that only a small percentage of BrS related mutations are studied functionally. We have performed an exhaustive compilation of reported mutations related to BrS located in the pore regions of Na v 1.5 channel (Table S1). Out of the 86 mutations identified in these regions, functional studies are documented only for 18 of them. The present work is the first reported functional study of a pore mutation in DII of Na v 1.5 associated with BrS.
Our electrophysiological studies in transfected HEK cells showed that the mutation caused a significant decrease in current density compared to WT. In addition, the activation curve of the I890T currents was shifted to more positive potentials. It has been previously shown that mutations in the pore region may affect voltage activation of Na v 1.5 and other Na v channels [32][33][34][35]. The hypothesis that mutations in the pore region of Na v channels can lead to structural perturbation compromising activation has been put forward [32]. In this context, our immunodetection experiments indicated that the decrease in I Na observed in I890T cells could not be explained by a reduction in the membrane expression of the channel. Collectively, our observations support the idea that functional changes observed in the I890T currents are likely due to altered intrinsic properties of the channel.
The advent of the crystal structure of Na v Ab [11] has provided a structural framework to model mutations in sodium channels. The homologous residue to I890Na v 1.5 is T169Na v Ab (Fig. 6A,  upper panel). This Thr is conserved in the NaChBac bacterial sodium channel (T187NaChBac) and in the sodium channel of alphaproteobacteria HIMB114 (T172Na v Rh), the structure of which has recently been solved [36]. In the available structure of Na v Ab channel, T169Na v Ab is buried within the intramembrane region of the pore. The polar group of T169Na v Ab is stabilized by a hydrogen bond to R185Na v Ab of the adjacent subunit [11]. The Na v Rh channel stabilizes T172 in a similar fashion: the alcohol group of T172Na v Rh is within hydrogen bond distance (2.7-3.2 Å depending on the subunit) of the carbonyl oxygens of R162 and I168 of the same chain. Interestingly, the mutation I890T recollects the bacterial residue at this position. However, in our I890T Na v 1.5 model, no hydrogen bond acceptor candidate lies in the proximity of T890 and we cannot predict how the introduction of this polar group might be stabilized. It is interesting to note that isoleucine is the aminoacid present in position 890 in Na v 1.5 as well as in the homologous position of different sodium channels in a wide variety of vertebrates ( Figure S1). Thus, although the precise role of I890 in Na v 1.5 is unknown, its presence at this position has been evolutionary favored.
In summary, we have identified a Na v 1.5 pore mutation, I890T, in a BrS patient. This novel mutation causes an evident reduction in I Na and a depolarizing shift in current voltage-dependent activation. Both mutation-dependent effects create the conditions for the observed pathophysiological manifestations of the patient. Although the observed changes in channel function in the mutated protein are mild, we cannot exclude that the effects of the mutation I890T in native myocytes could be different from those observed in our HEK cell experimental model, due to different regulatory factors. In addition, our functional study is analyzed in the context of the recently published crystal structure of the bacterial Na v Ab and Na v Rh channels. The evident, although mild, functional effect of the mutation correlates well with the lack of major structural changes found in the in silico analysis. In this sense, we believe that this type of studies hold the promise that correlations among channel structure, functional effects of mutations and clinical studies can lead to better understanding of SCD-related channelopathies.

Limitations of the Structural Model
The most important limitation of our modelling approach was the use of 2 bacterial sodium channel structures (Na v Ab and Na v Rh resolution 2.7 and 3.05 Å , respectively) to model the pore domain of DII in Na v 1.5. Amino acid sequences were only moderately conserved in S5-S6 of DII in Na v 1.5 compared to Na v Rh (17% identity and 25% homology) and Na v Ab (22% identity and 46% homology). Consequently, at the resolution of the model, it was not straight-forward to anticipate the precise structural role of I890 in Na v 1.5. We have tentatively proposed that the observed electrophysiological changes in I890T may be due to the introduction of the polar group of T890. This speculation was mostly based on the observation that T890 was stabilized by hydrogen bonds in bacterial channels, an interaction that was difficult to envision in I890T Na v 1.5 models. We acknowledge that this is an indirect argument. We believe, though, that our in silico analyses as well as the alignment data presented here support the idea that, in the absence of a neighboring hydrogen donor, an isoleucine may be more appropriate at that position. Figure S1 I890 is a highy conserved aminoacid among vertebrates. Sequence alignment of voltage-gated sodium channel a-subunit family members of different species. Human Na v 1.5 I890 and its homologues are marked with a dark box. Identical amimoacids are highlighted in grey. Similar aminoacids are included inside light boxes. (TIF)