Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8

Abstract

Background: Adenosine triphosphate (ATP)-sensitive potassium cardiac channels consist of inward-rectifying channel subunits Kir6.1 or Kir6.2 (encoded by KCNJ8 or KCNJ11) and the sulfonylurea receptor subunits SUR2A (encoded by ABCC9).

Objective: To examine the association of mutations in KCNJ8 with Brugada syndrome (BrS) and early repolarization syndrome (ERS) and to elucidate the mechanism underlying the gain of function of ATP-sensitive potassium channel current.

Methods: Direct sequencing of KCNJ8 and other candidate genes was performed on 204 BrS and ERS probands and family members. Whole-cell and inside-out patch-clamp methods were used to study mutated channels expressed in TSA201 cells.

Results: The same missense mutation, p.Ser422Leu (c.1265C>T) in KCNJ8, was identified in 3 BrS and 1 ERS probands but was absent in 430 alleles from ethnically matched healthy controls. Additional genetic variants included CACNB2b-D601E. Whole-cell patch-clamp studies showed a 2-fold gain of function of glibenclamide-sensitive ATP-sensitive potassium channel current when KCNJ8-S422L was coexpressed with SUR2A-wild type. Inside-out patch-clamp evaluation yielded a significantly greater half maximal inhibitory concentration for ATP in the mutant channels (785.5 ± 2 vs 38.4 ± 3 μM; n = 5; P <.01), pointing to incomplete closing of the ATP-sensitive potassium channels under normoxic conditions. Patients with a CACNB2b-D601E polymorphism displayed longer QT/corrected QT intervals, likely owing to their effect to induce an increase in L-type calcium channel current (I(Ca-L)).

Conclusions: Our results support the hypothesis that KCNJ8 is a susceptibility gene for BrS and ERS and point to S422L as a possible hotspot mutation. Our findings suggest that the S422L-induced gain of function in ATP-sensitive potassium channel current is due to reduced sensitivity to intracellular ATP.

Figure 1. Pedigrees of Brugada syndrome (BrS) and early repolarization syndrome (ERS) families

A: BrS family with atrial fibrillation (AF), sudden cardiac death (SCD) and sudden infant death syndrome (SIDS). B: BrS with AF. C: ERS with Aborted Sudden Cardiac Death (ASCD). D: BrS family. (+/−) indicates heterozygosity for KCNJ8-S422L and or CACNB2b-D601E. (−/−) indicates a negative genotype. Arrows denote the probands. Numbers represent MMRL ID of probands. Variable penetrance is due in part to age and gender of family members in the case of J wave syndromes.

Figure 2. Electrocardiograms (ECGs) of Brugada and early repolarization syndromes probands

A: 12-lead ECG of Brugada syndrome (BrS) proband (#747) recorded after ajmaline displaying ST segment elevation in V1, V2 and V3. B: BrS proband (#171) shows ST segment elevation after ajmaline challenge (left panel) and polymorphic ventricular tachycardia (VT) induced using 2 extrastimuli (240 ms and 170 ms) applied to the right ventricular apex (RVA, right panel). C: Early repolarization syndrome (ERS) (pedigree shown in Figure 1D) J wave elevation denoted by arrows in leads I, II, AVF, V4, V5 and V6 preceding initiation of monomorphic VT.

Figure 3. Genetic mutation in KCNJ8 gene

A: DNA chromatogram showing a heterozygous C-to-T transition at nucleotide 1265, predicating a substitution of a leucine (Leu) for serine (Ser) at residue 422 (S422L) of KCNJ8, which encodes the Kir6.1 subunit of the ATP-sensitive K (K_ATP) channel current (I_K-ATP). B: Predicted topology of the Kir6.1 and SUR2A subunit showing the location of the S422L mutation (red circle) at the carboxyl terminal region of Kir6.1; C: Functional K_ATP channels have an octameric subunit structure with four pore-forming subunits (Kir6.1) and four sulfonylurea receptors (SUR2A). D: Amino acid alignments performed using GenBank accession numbers corresponding to protein sequences shows a serine at position 422 is highly conserved among species.

Figure 4. Whole-cell patch clamp studies suggesting that KCNJ8-S422L is associated with a gain of function in ATP-sensitive K (K_ATP) channel current (I_K-ATP)

A: Current-voltage relations of I_K-ATP channel obtained 48–72 hours following transient transfection of human Kir6.1-wild type (WT) (KCNJ8-WT) and Kir6.1-S422L (KCNJ8-S422L) with human SUR2A-WT genes in TSA201 cells. Representative traces recorded before and after gliblenclamide (10 µM) were digitally subtracted to obtain the glibenclamide-sensitive K_ATP current. Macroscopic currents were recorded from a holding potential of −80 mV followed by ramp protocol from −100 mV to 100 mV for 400 ms (see insert). B: Summary of the difference in I_K-ATP between KCNJ8-WT (black bar) and KCNJ8-S422L (red bar) channels at 0 mV and +40 mV, respectively. Data points are the mean±SEM of peak whole-cell currents (n=8 cells for each group). *P<0.05 and **P<0.01 mutant vs WT.

Figure 5. KCNJ8-S422L mutation causes a gain of function in ATP-sensitive K (K_ATP) channel current (I_K-ATP) by reducing sensitivity of K_ATP channels to ATP

Sensitivity to K₂ATP of KCNJ8-S422L/SUR2A-wild type (WT) and KCNJ8-WT/SUR2A-WT channels measured using excised inside-out patches. A: I_K-ATP traces from excised inside-out macropatches, obtained by holding the patch at 0 mV and applying ramps from −80 to +80 mV (3 s), before and after attaining steady-state inhibition with K₂ATP. B: Concentration-response relationships. Currents were normalized to the current recorded at −80 mV under control conditions. The IC₅₀ value of K₂ATP for KCNJ8-WT/SUR2A-WT was 38.4±3 µM (n=5), whereas the IC₅₀ for the mutant KCNJ8-S422L/SUR2A-WT was 785.5±2 µM (n=5, p<0.01). Mean ±SEM. **P<0.01 vs. WT.

Figure 6. Modulatory effect of a secondary gene variant on QTc interval in probands with the S442L-KCNJ8 gain of function mutation

The S422L-KCNJ8 mutation alone is associated with a relatively short QTc interval. Probands with a second genetic variant causing an increase in late calcium channel current display a relatively longer QTc interval. The longest QTc is found in females in whom a gain of function genetic variant in CACNB2b is detected leading to an increase in late I_Ca.