Functional Characterization of Two Novel Mutations in SCN5A Associated with Brugada Syndrome Identified in Italian Patients

Figures

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  Figure 1. Pedigree and ECG of the BrS families. (A) Pedigree of family of patient I (III-1) carrying the missense variation c.3929C>T (Pro1310Leu) in SCN5A. The genetic analysis was performed only in proband’s son who resulted negative for the Pro1310Leu variation. I-1: SCD at 87 years; II-1: SCD at 78 years; III-2: SCD at 51 years. (B) ECG of patient I shows Brugada type 1 pattern in V1-V2 recorded at standard intercostal space. (C) Pedigree of family of patient II (II-1) carrying Gly1687_Ile1688insGlyArg in heterozygosis in SCN5A. Her son (III-1) carries both the maternal variation and the His558Arg benign polymorphism in SCN5A. The Ins1687GR insertion was searched exclusively in the two subjects manifesting symptoms and/or BrS ECG pattern, in light of the uncertain significance of the variation. I-1: SCD at 38 years; II-2: SCD at 50 years; III-2: syncope and negative drug-induced testing for BrS. (D) ECG of patient II shows Brugada type 1 pattern in V1-V2 recorded at second intercostal space.

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  Figure 2. Localization of the BrS mutations in the Nav1.5 channel modeled on the 3D structure of the rat Nav1.5 α-subunit (pdb: 6UZ3). Lateral (A) and top (B) view of Nav1.5 channel showing the P1310L mutation depicted in purple. Lateral (C) and top (D) view of the Nav1.5 channel showing the Ins1687GR insertion in purple. The DEKA residues are shown in red.

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  Figure 3. Current density of Nav1.5 WT, P1310L and WT+P1310L channels expressed in HEK 293 cells. (A) Representative sodium current traces from cells transfected with Nav1.5 WT (3 µg), P1310L (3 µg), and Nav1.5+P1310L (1.5 µg +1.5 µg) cDNAs. The voltage protocol is shown in the inset. (B) IV plot showing the mean current density of Nav1.5 WT, P1310L and Nav1.5+P1310L channels as a function of membrane potential. (C) Bar graph showing the mean current density measured at −30mV for the indicated channels. Data are mean ± SE; n = 11–20 cells. * p < 0.05 for P1310L channels compared with Nav1.5 WT.

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  Figure 4. Biophysical properties of Nav1.5 WT, P1310L, and WT+P1310L channels expressed in HEK 293 cells. (A) Voltage-dependence of activation. (B) Voltage-dependence of fast inactivation. (C) Recovery from fast inactivation. Voltage protocols are indicated in the insets and detailed in the Supplementary Materials and Methods. (D) Bar graph showing window current measure for the indicated channels. The inset shows the triangular area under the overlapping point of the activation and inactivation curves of the respective channels. (E) Bar graph showing the amplitude of the sustained current measured at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells.

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  Figure 5. Current density of Nav1.5WT, Ins1687GR, and WT+Ins1687GR channels expressed in HEK 293 cells. (A) Representative sodium current traces from cells transfected with Nav1.5 WT (3 µg), Ins1687GR (3 µg) and Nav1.5+Ins1687GR (1.5 µg +1.5 µg) cDNAs. The voltage protocol is shown in the inset. (B) IV plot showing the mean current density of Nav1.5 WT, Ins1687GR and WT+Ins1687GR channels as a function of membrane potential. (C) Bar graph showing the mean current density measured at -30mV for the indicated channels. Data are mean ± SE; n = 11–21 cells. * p < 0.05 for Ins1687GR channels compared with Nav1.5 WT.

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  Figure 6. Biophysical properties of Nav1.5WT and WT+Ins1687GR channels expressed in HEK 293 cells. (A) Voltage-dependence of activation. (B) Voltage-dependence of fast inactivation. (C) Recovery from fast inactivation. Voltage protocols are indicated in the insets and detailed in the Supplementary Materials and Methods. (D) Bar graph showing window current measure for the indicated channels. The inset shows the triangular area under the overlapping point of the activation and inactivation curves of the respective channels. (E) Bar graph showing the amplitude of the sustained current measured at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells.

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  Figure 7. Plasma membrane localization of Nav1.5 WT, P1310L and Ins1687GR channels. (A) Immunofluorescence confocal microscopy analysis of the Nav1.5 WT, P1310L and Ins1687GR channels (green signal; left) and plasma membrane marker WGA-555 (red signal, middle) in HEK 293 cells grown on coverslips. The overlay column reports the co-localization of the two fluorescence signals (yellow, right). WGA, Wheat Germ Agglutinin. The horizontal bar indicates 10 μm. (B) Immunoblotting analysis of Nav1.5 WT, Ins1687GR and P1310L channels expressed in HEK 293 cells. The position of molecular weight markers is at the right of the blots. Expressions of β-actin are displayed as controls for the loaded protein amounts. (C) Bar graph showing the total protein expression level for the indicated channel. For total protein expression, density was standardized as the ratio of the Nav1.5 signal to the cognate β-actin signal. Quantitative analysis was performed from 3 independent experiments.

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  Figure 8. Effect of 24h incubation with 300 μM mexiletine on Nav1.5 WT and Ins1687GR channels. (A) Representative sodium current traces from cells expressing Nav1.5 WT and Ins1687GR channels in control solution and after 24 h incubation with 300 μM mexiletine. Mexiletine, was washed out prior to recording sodium currents. (B) IV plot showing the mean current density of Nav1.5 WT and Ins1687GR channels as a function of membrane potential before and after 300 μM mexiletine incubation. (C) Voltage-dependence of activation for Nav1.5 WT and Ins1687GR channels in control solution and after 300 μM mexiletine incubation. (D) Voltage-dependence of fast inactivation for Nav1.5 WT and Ins1687GR channels in control solution and after 300 μM mexiletine incubation. (E) Recovery from fast inactivation for Nav1.5 WT and Ins1687GR channels before and after 300 μM mexiletine incubation. Data are mean ± SE; n = 6–8 cells.