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

Figures

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  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

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  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

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  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 Ito 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 Ito 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

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  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