Brugada syndrome associated with SCN5A E1053K mutation. (A) ECG monitor strip of the index patient carrier of the E1053K mutation showing self-terminating polymorphic ventricular tachycardia recorded at the arrival to the emergency room. (B) ECG recordings in leads V1–V3 of the index patient carrier of the E1053K mutation at baseline and during i.v. flecainide administration (2 mg/kg in 10 min). (C) Human Na_v1.5 proposed secondary structure. Potential ankyrin-binding motif is located in loop 2. Human Brugada mutation E1053K is located within these nine amino acids.

WT Na_v1.5 and Na_v1.5 E1053K voltage dependence of activation and kinetics of inactivation. (A) Averaged and normalized current traces recorded from WT (n = 33) and E1053K mutant channel (n = 18) obtained by applying depolarizing steps (–45 mV, –40 mV, –35 mV, –20 mV, –15 mV) for 25 ms. (B) Normalized current–voltage relationship for WT and E1053K recorded in 10 mM Na⁺ (n = 5 for WT, n = 5 for E1053K). The protocol is shown in the Inset. (C) Steady-state activation curve obtained by normalizing the peak current for the conductance over a range of voltages from –100 mV to 15 mV (n = 5 for WT and n = 5 for E1053K). Data are fitted with a Boltzmann function. (D) Kinetics of the onset of inactivation measured as time to half-inactivation. Given t₁ as the time to the maximum peak and t₂ as the time to 50% of current inactivation (*), t_h = (t₂ – t₁). See Inset for protocol.

Human SCN5A mutation E1053K eliminates interaction with ankyrin-G. (A) Alignment of ankyrin-binding motif from Na_v1.2 with other Na_v isoforms. Bottom is Na_v1.5 amino acid alignment from human with Brugada syndrome (E1053K). (B) NF/Na_v1.5 chimeras. A diagram of NF/Na_v1.5 chimeras using loop 2 sequence from Na_v1.5, Na_v1.5Δ (deletion of VPIAVAESD), or Na_v1.5 E1053K mutant is shown. Constructs were expressed in baby hamster kidney cells and lysates incubated with GST-AnkG MBD(+)- or GST(–)-conjugated beads. Bound protein (Left) or 6% of total lysate used (Right) was analyzed by immunoblotting with HA-specific antibodies. The doublet observed in immunoblots represents both glycosylated and nonglycosylated forms of NF/Na_v1.5 chimeras in total cell lysates (23). (C) Saccharomyces cerevisiae was cotransformed with pBTM116-Na_v1.5, pBTM116-Na_v1.5Δ, or pBTM116-Na_v1.5 E1053K, and pGAD424 alone, and pGAD424-MBD. Cotransformants were selected on –LT media and then tested for HIS3 reporter gene activation. (D) 190-kDa ankyrin-G associates with Na_v1.5 in vivo. Lane 1, Coomassie blue stained gel of rat heart lysate. Lane 2, immunoblot analysis of lane 1 by using ankyrin-G polyclonal Ig. (E) Adult rat ventricle detergent-soluble lysate was incubated with affinity-purified ankyrin-G Ig conjugated to beads or control Ig alone. Bound protein was eluted and analyzed by immunoblot analysis using Na_v1.5-specific antibody (SH1). Input, 20% of soluble protein.

One hundred ninety-kilodalton ankyrin-G is coexpressed with Na_v1.5 at intercalated disc and T-tubule membranes and Na_v1.5 E1053K mutant is mislocalized from intercalated disc/T-tubule membrane of cultured adult rat cardiomyocytes. (A) Ankyrin-G localization (green) at intercalated disc and T-tubule membranes of adult rat cardiomyocytes colabeled with N-cadherin (red). (Scale bar, 10 μm.) (B) Na_v1.5 localization (green, SH1 antibody) at intercalated disc and T-tubule membranes of adult rat cardiomyocytes colabeled with N-cadherin (red). (Scale bar, 10 μm.) (C) Localization of N-cadherin (red) and lentiviral-expressed WT HA-Na_v1.5 (green, HA antibody) in 4-day cultured adult rat cardiomyocytes. (D) Localization of N-cadherin (red) and lentiviral-expressed WT HA-Na_v1.5 E1053K (green, HA antibody) in 4-day-old adult rat cardiomyocytes. (E–G) High-magnification image of intercalated disk region of above images. Intercalated disc marked by arrowhead. T-tubules are denoted by yellow arrows. Note small puncta near T-tubules in HA Na_v1.5 E1053K-transduced cell. (Scale bar, 10 μm.) (H) Localization of N-cadherin (red) and lentiviral-expressed WT HA-Na_v1.5 (green, HA antibody) in nonpermeabilized 4-day cultured adult rat cardiomyocytes. Intercalated disc is marked by arrowhead. T-tubules are denoted by yellow arrows. (I) Localization of N-cadherin (red) and lentiviral-expressed WT HA-Na_v1.5 E1053K in nonpermeabilized 4-day cultured adult rat cardiomyocytes. (Scale bar for H and I, 10 μm.)

Na_v1.5 E1053K displays abnormal inactivation properties compared to WT Na_v1.5. (A) The transition in the close-state inactivation is favored for the E1053K mutant channel respect to WT (n = 13 for WT, n = 11 for E1053K). (B) The recovery from fast inactivation is slower in the E1053K channel compared to the WT channel (n = 9 for WT, n = 6 for E1053K). (C) Enhancement of the intermediate inactivation state for Na_v1.5 E1053K compared to WT Na_v1.5 (n = 10 for WT, n = 5 for E1053K). After 200 ms, there is 64% available for E1053K vs. 83% of the WT. In each panel, see Inset for protocol.