Transient outward current (I(to)) gain-of-function mutations in the KCND3-encoded Kv4.3 potassium channel and Brugada syndrome

Abstract

Background: Brugada syndrome (BrS) is a sudden death-predisposing genetic condition characterized electrocardiographically by ST segment elevation in the leads V(1)-V(3). Given the prominent role of the transient outward current (I(to)) in BrS pathogenesis, we hypothesized that rare gain-of-function mutations in KCND3 may serve as a pathogenic substrate for BrS.

Methods: Comprehensive mutational analysis of KCND3-encoded Kv4.3 (I(to)) was conducted using polymerase chain reaction, denaturing high performance liquid chromatography, and direct sequencing of DNA derived from 86 unrelated BrS1-8 genotype-negative BrS patients. DNA from 780 healthy individuals was examined to assess allelic frequency for nonsynonymous variants. Putative BrS-associated Kv4.3 mutations were engineered and coexpressed with wild-type KChIP2 in HEK293 cells. Wild-type and mutant I(to) ion currents were recorded using whole-cell patch clamp.

Results: Two BrS1-8 genotype-negative cases possessed novel Kv4.3 missense mutations. Both Kv4.3-L450F and Kv4.3-G600R were absent in 1,560 reference alleles and involved residues highly conserved across species. Both Kv4.3-L450F and Kv4.3-G600R demonstrated a gain-of-function phenotype, increasing peak I(to) current density by 146.2% (n = 15, P <.05) and 50.4% (n = 15, P <.05), respectively. Simulations using a Luo-Rudy II action potential (AP) model demonstrated the stable loss of the AP dome as a result of the increased I(to) maximal conductance associated with the heterozygous expression of either L450F or G600R.

Conclusions: These findings provide the first molecular and functional evidence implicating novel KCND3 gain-of-function mutations in the pathogenesis and phenotypic expression of BrS, with the potential for a lethal arrhythmia being precipitated by a genetically enhanced I(to) current gradient within the right ventricle where KCND3 expression is the highest.

Figure 1. Identification of KCND3-L450F and KCND3-G600R Mutations in BrS

Mutations in KCND3 are associated with Brugada syndrome. Depicted are A, DHPLC profiles (normal, red trace and abnormal, green trace) and B, DNA sequencing chromatograms. C, Predicted protein topology schematic of Kv4.3 indicating the location of the L450F and G600R mutations. D, Sequence conservation across species for the L450F and G600R mutations.

Figure 2. Representative Electrocardiograms for the L450F- and G600R-Positive BrS Index Cases

A, Representative basal Holter ECG demonstrating spontaneous ST-segment elevation in leads V₂ and V₃ and flecainide challenge twelve-lead ECG which unmasked a coved-type BrS ECG pattern in the L450F-positive BrS index case. B, Representative baseline twelve-lead ECG of the G600R-positive BrS index case with V₁ through V₃ leads in the normal position.

Figure 3. Kv4.3-L450F and Kv4.3-G600R Plus KChIP2 Increase I_to Current in Heterologous Cells

A, Representative whole-cell Kv4.3-WT plus KChIP2-WT (left), Kv4.3-L450F plus KChIP2-WT (middle) and Kv4.3-G600R plus KChIP2-WT (right) traces recorded in HEK293 cells elicited by step depolarization of 500 ms duration to +40 mV from a holding potential of −80 mV in 10 mV increments. B, The current voltage relationship for WT (n= 18), L450F (n=15) and G600R (n=15) Kv4.3 channels co-expressed with KChIP2-WT. All values represent mean±SEM. * p<0.05 vs. Kv4.3-WT plus KChIP2-WT. C. Bar graph showing peak current density at 40 mV for WT (n= 18), L450F (n=15) and G600R (n=15) Kv4.3 channels co-expressed with KChIP2-WT. * p<0.05 vs. Kv4.3-WT plus KChIP2-WT.

Figure 4. Kinetic Alteration for Kv4.3-WT, Kv4.3-L450F, or Kv4.3-G600R plus KChIP2

A, Inactivation time constants (τ) for Kv4.3-WT, Kv4.3–L450F, or Kv4.3–G600R plus KChIP2 currents plotted as a function of voltage. Inactivation time constants for each voltage step were determined by fitting a monoexponential function to current decay. All values represent mean±SEM. * p<0.05 vs. Kv4.3-WT plus KChIP2. B, Total charge of Kv4.3-WT, Kv4.3-L450F, or Kv4.3–G600R with KChIP2 as a function of voltage obtained by measuring the area under curve during the first 50 ms of each voltage step. * p<0.05 vs. Kv4.3-WT plus KChIP2. C, Steady-state inactivation curves of Kv4.3-WT, Kv4.3-L450F, or Kv4.3–G600R with KChIP2 determined from a holding potential of −100 mV to pre-pulse of −5 mV in 5 mV increments with 0.5 s duration followed by a test pulse of +20 mV with 0.5 s duration and fitted with a Boltzmann function. D, Recovery from inactivation of Kv4.3-WT, Kv4.3-L450F, or Kv4.3-G600R with KChIP2 determined from a holding potential of −80 mV to pre-pulse of +20 mV with 0.5s duration, with increased recovery interval, followed by a test pulse of +20 mV with 500 ms duration and fitted with a one-exponential function. All values shown represent Mean ±SEM.

Figure 5. Effects of Kv4.3-L450F and Kv4.3-G600R on the Right Ventricular Epicardial Action Potential

A, Simulated I_to current traces during step depolarization for 100 ms to 0 mV from a holding potential of −90 mV. Only first 60 ms are shown. Solid line – WT; dotted line – L450F; dashed line – G600R. B, RV epicardial action potentials simulated using WT, L450F, and G600R mutant I_to incorporated into a modified Luo-Rudy II AP model. BCL = 800 ms (75 bpm). Solid line – WT; dotted line – L450F; dashed line – G600R. Only the 5th AP in the equilibration chain is displayed here, the AP shape does not change in subsequent cycles.