Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes

Abstract

Background: J-wave syndromes have emerged conceptually to encompass the pleiotropic expression of J-point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). KCNJ8, which encodes the cardiac K(ATP) Kir6.1 channel, recently has been implicated in ERS following identification of the functionally uncharacterized missense mutation S422L.

Objective: The purpose of this study was to further explore KCNJ8 as a novel susceptibility gene for J-wave syndromes.

Methods: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J-wave syndromes, including 87 with BrS and 14 with ERS. Six hundred healthy individuals were examined to assess the allelic frequency for all variants detected. KCNJ8 mutation(s) was engineered by site-directed mutagenesis and coexpressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole-cell configuration of the patch-clamp technique.

Results: One BrS case and one ERS case hosted the identical missense mutation S422L, which was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1,200 reference alleles. Both cases were negative for mutations in all known BrS and ERS susceptibility genes. K(ATP) current of the Kir6.1-S422L mutation was increased significantly over the voltage range from 0 to 40 mV compared to Kir6.1-WT channels (n = 16-21; P <.05).

Conclusion: These findings further implicate KCNJ8 as a novel J-wave syndrome susceptibility gene and a marked gain of function in the cardiac K(ATP) Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS.

Figure 1. Molecular elucidation of KCNJ8-S422L

A. Top panel, the proband’s DHPLC profile and DNA sequence chromatogram showing a C>T substitution at position 1265 of KCNJ8 resulting in a Serine (S) to Leucine (L) substitution at position 422 (S422L). Bottom panel, the normal DHPLC profile and DNA sequence chromatogram. B. Conservation across species for S422 in Kir6.1 C. Linear topology of the Kir6.1/SUR2 complex showing the predicted localization of the S422L missense mutation to the C-terminus. SUR2= Sulfonylurea receptor type 2.

Figure 2. Case 1 Electrocardiographic Phenotype

12-lead ECGs of case 1 showing intraventricular conduction delay (V1), important dynamic repolarization abnormalities in leads V1-V4 (A, B) and intermittent J waves in the inferior leads (A,B,D). C. ICD electrogram of VT- therapy with both atrial electrogram (top) and ventricular electrogram (bottom). The VT episode is unsuccessfully shocked; the arrhythmia was self-terminated before the second ICD therapy (electrogram not shown). D. Magnification of the “J” wave observed in lead DIII from the 12-lead ECG shown in panel B.

Figure 3. Case 2 Electrocardiographic Phenotype

A. Flecainide challenge unmasked the typical coved type Brugada Syndrome ECG pattern in case 2. B. Spontaneous type 1 Brugada ECG pattern recorded during 24 hour Holter monitoring in case 2.

Figure 4. Marked gain-of-function for Kir6.1-S422L channels

Comparing the glibenclamide (Glib)-sensitive K_ATP current of Kir6.1-S422L with Kir6.1-WT. (A) Examples of whole cell current traces of Kir6.1-WT recorded after 1-2 minutes perfusion of 100 μM pinacidil (maximal I_k, left), in the presence of 20 μM Glib (middle), and the Glib-sensitive I_KATP obtained by subtraction (right). (B) Examples of whole cell current traces of Kir6.1-S422L recorded after 1-2 minutes perfusion of 100 μM pinacidil (maximal I_k, left), in the presence of 20 μM Glib (middle), and the Glib-sensitive I_KATP obtained by subtraction (right). (C) Summary data of the current voltage plot for the Glib-sensitive I_KATP at the voltages tested (insert, n=16-21 cells. * p<0.05). (D) Bar graphs showing the mean I_KATP densities from Kir6.1-WT and Kir6.1-S422L channels elicited by a test pulse from holding potential of -40 mV to 40 mV for 260 ms. I_KATP density of Kir6.1-S422L mutant channels was increased significantly compared with Kir6.1-WT channels.