Unravelling Novel SCN5A Mutations Linked to Brugada Syndrome: Functional, Structural, and Genetic Insights

Figure 1. Pedigrees of the three unrelated families. (A) Family 1, (B) Family 2, and (C) Family 3. Arrows indicate the proband in each family. Squares: male; circles: female; deceased individuals are indicated with a slash; BrS individuals are indicated with black filled symbols. Reported are also the proband’s sequence identification of the base substitution and her/his ECG in the insets. Only in the proband of family 1 was the BrS pattern drug-induced; in families 2 and 3, the proband’s ECG type 1 BrS pattern was spontaneous (for complete ECGs, see Figure S1 in the Supplementary Materials).

Figure 2. Electrophysiological characterization of Nav1.5 constructs. Representative current traces and their biophysical properties measured via the patch clamp approach in HEK293 cells overexpressing the constructs as indicated: WT in black (A), WT/p.A344S or p.A344S in blue (B), WT/p.N347K or p.N347K in green (C), and WT/p. D349N or p.D349N in red (D). Panels B, C, and D present from left to right: families of current traces, current–voltage relationship showing the reduction in the mean current density recorded in the presence of the mutations in heterozygous or homozygous conditions with respect to the WT channel, fitting the data with the Boltzmann equation (see Section 4) provided midpoint activation and inactivation potential reported in Table 1, recovery from inactivation (see Table 1 for the kinetic parameters), and the kinetics of the development of the intermediate inactivation. Data are shown as mean ± SEM.

Figure 3. Localization of Nav1.5 WT and mutants transiently transfected in HEK293 cells. Representative immunofluorescence confocal images showing the plasma membrane and cytosolic localization of WT Nav1.5 protein (A), along with its p.A344S (B), p.N347K (C), and p.D349D (D) mutants. Nav1.5 is labeled in green. Scale bar: 10 µm.

Figure 4. Effect of mexiletine on the mutated constructs. (A) Representative current traces of mutated channels recorded after mexiletine incubation in response to a depolarizing stimulus at −20 mV in HEK293 cells expressing the constructs as indicated: WT in black (for reference), p.A344S in blue, p.N347K in green, and p.D349N in red. (B). Scattered plot showing the distribution of the current density values obtained in absence (filled symbols) and in presence (empty symbols) of mexiletine. The black line represents the average values (see Table 1). (C) Fitting the data with the Boltzmann equation (see the Section 4) provided midpoint activation potential, as reported in Table 1. Data are shown as mean ± SEM.

Figure 5. Structural analysis and mapping of the Nav1.5 mutations. (A) At the top, structural mapping of the Nav1.5 mutations reported in this study based on their cryo-EM structure (PDB id: 7DTC); on the bottom, a left extracellular view of Nav1.5 electrostatic potentials calculated with the APBS method and close-up of Eα1 on the bottom right. Interactions between p.A344 (B), p.N347, (C) and p.D349 (D) in WT Nav1.5. The Cα atoms of these residues are shown in magenta. The SF-supporting helices P1 and P2 are depicted in blue and yellow, respectively. The DEKA residues are shown as orange spheres. (E) Multiple sequence alignment of SCN5A genes in Eukaryota and (F) multiple sequence alignment of human SCN genes with their isoforms. Black stars indicate the mutation sites presented in this study. The selectivity filter (SF)-supporting pore helices P1 and P2 regions are lined above the sequence in black, and the residue p.D372, belonging to the DEKA motif (SF), is marked with a black dot.