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 (I_to ), is associated with early repolarization syndrome (ERS). Previous studies showed that mutations in SCN1Bβ both to increase the I_to 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 (I_Na ) when co-expressed with SCN5A/WT. Co-expression of KCND3/WT and SCN1Bβ/S248R or SCN1Bβ/R250T increased the transient outward potassium current I_to 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 I_to 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 I_to by associated with Kv4.3, which maybe underlie the ERS phenotype of the probands.

Keywords: SCN1Bβ; early repolarization syndrome; transient outward potassium current.

Figure 1

Genetic analyses and ECG in four families with early repolarization syndrome. A, Four family pedigrees with early repolarization syndrome (ERS). Black symbol: early repolarization pattern with ventricular fibrillation events. Grey symbol: early repolarization pattern without ventricular fibrillation events. B, Twelve‐lead ECG in a 14‐year‐old boy in Family 1 (arrow). C, DNA sequencing traces (chromatograms) for S248R and R250T variants identified in SCN1Bβ

Figure 2

Sodium Currents recorded on HEK293 cells co‐expressed of SCN5A/WT and SCN1Bβ. A‐C, macroscopic currents recorded at Vm in the range −120 to +80 mV from a holding potential of −120 mV. Protocol used was shown in inset. D, Current‐voltage (I‐V) relationship of WT, S248R and R250T. (n = 9, 13 and 14, respectively, *P < 0.05 vs WT). E, Voltage dependent steady‐state activation (V _1/2 = −54.10 ± 1.06 mV, n = 7 for WT; −51.81 ± 0.99 mV, n = 9 for S248R; −53.79 ± 1.02 mV, n = 6 for R250T). F: Voltage dependent steady‐state inactivation (V _1/2 = −72.62 ± 0.64 mV, n = 9 for WT; −75.94 ± 0.62 mV, n = 13 for S248R; −75.04 ± 0.37 mV, n = 12 for R250T). E and F plots were fitted by Boltzmann equation: y = 1/[1 + exp(V−V _1/2)/k], V _1/2 = voltage at which sodium current is half‐maximally activated, k = slope factor. G, Recovery from inactivation (n = 13, 14 and 14 for WT, S248R and R250T, respectively). Protocol used was shown in inset. The currents recorded at P2 were normalized to that at P1. Two‐exponential equation was used to fit the plot

Figure 3

Transient outward potassium current recorded on HEK293 cells co‐expressed of KCND3/WT and SCN1Bβ. A‐C, macroscopic currents recorded at Vm in the range −80 to +80 mV from a holding potential of −80 mV. Protocol used was shown upside. D, Current‐voltage (I‐V) relationship of WT, S248R and R250T. (n = 9, 8 and 12, respectively. *P < 0.05 vs WT. **P < 0.01 vs WT). E, the mean peak I_to current densities recorded on repolarization to −80 mV from each group. *P < 0.05 vs WT; **P < 0.01 vs WT. F, Voltage dependent steady‐state activation (V _1/2 = 26.58 ± 1.15 mV, n = 9 for WT; 28.29 ± 1.05 mV, n = 7 for S248R; 28.24 ± 1.04 mV, n = 12 for R250T). G, Voltage dependent steady‐state inactivation (V _1/2 = −49.52 ± 0.41 mV, n = 9 for WT; −39.25 ± 0.61 mV, n = 8 for S248R; −40.39 ± 0.59 mV, n = 12 for R250T). F and G plots were fitted by Boltzmann equation: y = 1/[1 + exp(V−V _1/2)/k], V _1/2: voltage at which I_to is half‐maximally activated, k = slope factor. H, Recovery from inactivation (n = 8, 6 and 10 for WT, S248R and R250T respectively). Protocol used was shown in inset. The currents recorded at P2 were normalized to that at P1. Two‐exponential equation was used to fit the plot

Figure 4

Co‐immunoprecipitation study indicated direct interaction of KCND3 and SCN1Bβ subunits. HEK293 cells were co‐transfected with KCND3/WT and SCN1Bβ (WT, S248R, R250T). Cells were lysed and total protein extracts were immunoprecipitated using anti‐KCND3 and then immunoblot with anti‐KCND3 and anti‐SCN1Bβ. A: Representative western blots of KCND3 (75 kD arrow) and SCN1Bβ (30 kD arrow). IP: immunoprecipitated pellet; SN: supernatant. B: Percentage of SCN1Bβ (WT, S248R, R250T) co‐immunoprecipitation related to the total amount of KCND3/WT immunoprecipitated. *P < 0.05 vsWT