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SCN1Bβ mutations that affect their association with Kv4.3 underlie early repolarization syndrome

Abstract Background Abnormal cardiac ion channels current, including transient outward potassium current (Ito), is associated with early repolarization syndrome (ERS). Previous studies showed that mutations in SCN1Bβ both to increase the Ito current and to decrease the sodium current. Yet its role in ERS remains unknown. Objective To determine the role of mutations in the SCN1Bβ subunits in ERS. Methods We screened for mutations in the SCN1B genes from four families with ERS. Wild‐type and mutant SCN1Bβ genes were co‐expressed with wild‐type KCND3 in human embryonic kidney cells (HEK293). Whole‐cell patch‐clamp technique and co‐immunoprecipitation were used to study the electrophysiological properties and explore the underlying mechanisms. Results S248R and R250T mutations in SCN1Bβ were detected in 4 families’ probands. Neither S248R nor R250T mutation had significant influence on the sodium channel current density (IN a) when co‐expressed with SCN5A/WT. Co‐expression of KCND3/WT and SCN1Bβ/S248R or SCN1Bβ/R250T increased the transient outward potassium current Ito by 27.44% and 199.89%, respectively (P < 0.05 and P < 0.01, respectively) when compared with SCN1Bβ/WT. Electrophysiological properties showed that S248R and R250T mutations decreased the steady‐state inactivation and recovery from inactivation of Ito channel. Co‐immunoprecipitation study demonstrated an increased association between SCN1Bβ mutations and Kv4.3 compared with SCN1Bβ/WT (P < 0.05 and P < 0.01, respectively). Conclusion The S248R and R250T mutations of SCN1Bβ gene caused gain‐of‐function of Ito by associated with Kv4.3, which maybe underlie the ERS phenotype of the probands.


| INTRODUCTION
The early repolarization pattern (ERP) is characterized by an elevation of J-wave >0.1 mV and sometimes involving an ST-segment elevation in at least two contiguous leads. Recent studies have revealed that ERP is associated with a higher risk of malignant ventricular arrhythmia and sudden cardiac death (SCD). [1][2][3][4][5] When a subject with ERP and malignant ventricular arrhythmias, that often known as early repolarization syndrome (ERS). Since the high prevalence of ERP in the general population (1.3%-9.2%), [6][7][8][9] especially in younger physically active individuals 10,11 and male sex, 12 it is significant to determine whose individuals with such common electrophysiological pattern are at risk of sudden cardiac death.
Some studies revealed that cardiac ion channel mutations play a major role in the pathogenesis of malignant ERPs. Mutations in the inward-rectifier ATP-dependent K+ channel current (I KATP ), [13][14][15] L-type calcium current (I CaL ) 16,17 and transient outward potassium current (I to ) 18,19 can lead to ERPs. In inherited families, mutations in seven different genes (KCNJ8, CACNA1C, KCND3, KChip2, SCN5A, ABCC9, Ankyrin-2) have been associated with ERPs. [13][14][15][16][17][20][21][22][23] The transient outward potassium channel (I to ) is a multi-subunit protein complex comprised of pore-forming α-subunits and auxiliary β-subunits. 24 In humans, Kv4.3 encodes the α-subunits of I to. 25 In human ventricular cells, I to currents mediate the early phase of action potential repolarization, 18 which can reduce the action potential duration by accelerating the early repolarization velocity and progressively suppressing the voltage of plateau phase. 19 Increasing of I to currents in region during initial ventricular repolarization can result in a J-wave on the ECG. 26 Therefore, disorders in I to currents might be underlying mechanism of ERS.
SCN1B encodes the cardiac sodium channel β-subunit. 27 It is comprised of large extracellular immunoglobulin-like domains, a single transmembrane-spanning segment, and intracellular C-terminal domains. SCN1B has two kinds of transcripts, SCN1B and SCN1Bβ, which encodes Navβ1 and Navβ1b, respectively. 27 Functional analysis indicated that β-subunit encodes by SCN1B involved in modulation of sodium channel gating and voltage dependence, 28,29 expression of sodium channel at the cell surface, 30 and cell adhesion. 31 Here, we reported a mutation in the SCN1Bβ gene, which encodes the regulatory β-subunits of the transient outward potassium current (I to ), identified in four families with ERS. We studied whether mutant in the SCN1Bβ was associated with ERS and explore the possible mechanisms. Electrophysiology modification by SCN1Bβ mutant was evaluated using patch-clamp technology.

| Genetic analysis
Genomic DNA used for genetic analysis was isolated from peripheral blood samples. The protein coding sequences of the SCN1B genes were amplified by polymerase chain reaction (PCR) and directed sequenced. The DNA sequence was then compared with the reference sequence of NM_001037 (SCN1B isoform a), NM_199037 (SCN1B isoform b) and NM_001321605 (SCN1B isoform c).

| Electrophysiology
Electrophysiological studies were performed 48 hours after transfection. Membrane currents were recorded by whole-cell patch- 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%.
I Na currents were elicited by depolarizing pulses ranging from −90 mV to +40 mV in 10 mV increments with a holding potential (HP) at −120 mV. Peak currents were measured and I Na densities (pA/pF) were obtained by dividing the peak I Na by the cell capaci- I to currents were elicited from a HP of −80 mV with depolarizing voltage pulses from −80 mV to +80 mV for 400 ms. Current density (pA/pF) was calculated from the ratio of current amplitude to cell capacitance. Peak currents were normalized to the maximum peak I to amplitude. Normalized activation and inactivation curves were fit with a Boltzmann equation: the voltage at which sodium current is half-maximally activated, and k was the slope factor. Recovery from inactivation was assessed by a standard paired pulse protocol: a 500 ms test pulse to +50 mV (P1) was followed by a variable recovery interval at 380 mV, then by a second test pulse to +50 mV (P2). The plot of P2/P1 was then fit with two exponential to determine the time constants for recovery, using the equation:

| Statistical analysis
Data were presented as Mean ± SEM. Statistical comparisons were analysed using two-tailed Student's t-test and ANOVA with Student-Newman-Keuls test. A P value less than 0.05 was considered statistically significant.

| Clinical data and genetic analysis
Clinical data of the four families was showed in Table 1. Four family pedigrees with ERS were showed in Figure 1A. Figure 1B showed a 12-lead ECG of a 14-year-old boy from Family 1 (arrow in Figure 1A). Screening of SCN1Bβ in the four families revealed four mutations. Two were in UTR three of SCN1Bβ and two (c.C744A and c.G749C) were non-synonymous mutation in exon three. Polymerase chain reaction (PCR) based sequencing analysis revealed a C-to-A replacement at nucleotide 744 and a G-to-C replacement at nucleotide 749 ( Figure 1C), which result in a serine (S) to arginine (R) at residue 248 (S248R) and an arginine (R) to threonine (T) at residue 250 (R250T).

| Electrophysiological characterization of
SCN5A co-expressed with SCN1Bβ/WT, SCN1Bβ/ S248R and SCN1Bβ/R250T In order to assess the effects of S248R and R250T mutation on    Figure 3G). Figure 3H showed

| Co-IP study
To test whether SCN1Bβ has some direct effects on Kv4.3, we next used Co-IP to assess the relationship. KCND3/WT was co-expressed with SCN1Bβ/WT, SCN1Bβ/S248R or SCN1Bβ/R250T in HEK293 cells and isolated by pull-down using an antibody to Kv4.3. Figure 4A showed the association between Kv4.3 (~75 kD, top) and SCN1Bβ (~30 kD, bottom) when co-expressed. Compared with KCND3/WT + SCN1Bβ/WT, co-expressed with SCN1Bβ/R250T resulted in a significant increase of SCN1Bβ to Kv4.3. However, the amount of SCN1Bβ interact with Kv4.3 was not significant different between WT and S248R ( Figure 4B).   Values are Mean ± SEM, V 1/2 : voltage of half-maximally activated or inactivation, k: slope factor. *P < 0.01 vs WT.
The currents recorded at P2 were normalized to that at P1. Two-exponential equation was used to fit the plot T A B L E 3 Gating kinetics parameters of I to in HEK293 cells co-expressed of KCND3/WT and SCN1Bβ Groups Activation Inactivation Recovery I to plays an important role in the early repolarization phase and abbreviates action potential duration. 19 Inhibition of I to exerts an ameliorative effect in the setting of ERS by producing an inward shift in the balance of current during the early phases of the epicardial action potential. 32 Our results showed that S248R and R250T mutations in SCN1Bβ could increase I to channel activities when coexpressed with KCND3. As both of the mutations of SCN1Bβ we described resulted in an increase of I to channel current, we recognized that these mutations might contribute to ERS. However, whether the degree of augment in I to current is correlate with the clinical presentation and prognosis of ERS is still unknown.
SCN1B gene encodes sodium channel beta1 subunit (Na v β1) and sodium channel beta1b subunit (Na v β1b), which serves as auxiliary sub- In the present study, our results showed that co-expressed with SCN1Bβ/S248R or SCN1Bβ/R250T increased I to current density by slowing down steady-state inactivation and accelerating recovery from inactivation compared with co-expressed with SCN1Bβ/WT as previous study. However, no significant sodium current density was observed in this study thought some minor changes in channel gating kinetics. The probands included in our study showed slurred or notched J-wave on surfaced electrocardiogram. One of underlying mechanism of J-wave is prominent voltage gradients in early repolarization between endocardium and epicardium, 36 which partly due to significant downregulation of I to in endocardium in comparison with that of midmyocardium and epicardium. 37 Thus, gain-of-function mutation of I to channel current in epicardium was expected to produce similar voltage gradients and J-wave.
The effects of SCN1Bβ variants on Na v 1.5 currents had been described earlier. Some studied reported that SCN1Bβ variants reduced sodium currents by accelerating recovery from inactivation and decreasing the slow inactivation rate. 29,38 In contrast, the mutations we reported in the present study could not alter Nav1.5 channel currents significantly. We have noted a previously study identified the same mutations of SCN1Bβ in three asymptomatic members from a family with Brugada syndrome and sick sinus syndrome. 39 Together, it suggested that the S248R and R250T mutations of SCN1Bβ maybe not pathogenic to Na v 1.5 function.
Both of the mutations, S248R and R250T, located at the C-terminal of SCN1Bβ. This hydrophobic region included residues from 243 to 262, which serve as a transmembrane domain. 40 To date, few mutations in this area were characterized by functional analysis. In present study, we observed an influence in steady-state inactivation and recovery from inactivation of I to currents. Previously study reported that co-expressed with SCN1Bβ/WT slowed recovery of I to from inactivation when compared with that of KCND3/WT alone. 35 Besides, Co-IP study showed an impaired interaction between KCND3 and SCN1Bβ/R250T. Therefore, we hypothesis that mutation in this area may affect association between KCND3 and SCN1Bβ, either in direct or indirect manners.
In summary, we found that the S248R and R250T mutations in SCN1Bβ gene causes gain-of-function of I to by associated with Kv4.3. Our findings suggested that the mutations maybe underlie the ERS phenotype of the probands, and SCN1Bβ maybe one of the possible modulatory genes associated to ERS.