Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation

Abstract

Background: Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs.

Objective: The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF.

Methods: One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5.

Results: A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT.

Conclusion: The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.

Figure 1

Mutation screening of KCNE5. A: DNA sequence chromatogram showing the wild type (WT) and the corresponding amino acid sequence. B: DNA sequence chromatogram showing the heterozygous mutation (in bold) and the corresponding amino acid sequence. A heterozygous substitution of T for C in nucleotide 193 predicts substitution of phenylalanine (TTC) for leucine (CTC) in codon 65 (L65F). C: ECG of the proband recorded during normal sinus rhythm between episodes of atrial fibrillation.

Figure 2

Representative current trace recorded from I_Ks/CHO cells transiently transfected with KCNE5-WT. A: Current from stable cell line of I_Ks/CHO. B: Current from I_Ks/CHO transfected with 0.15 μg KCNE5-WT. C: Current from I_Ks/CHO transfected with 0.3 μg KCNE5-WT. D: Current from I_Ks/CHO transfected with 1.5 μg KCNE5-WT. Pulse protocol is shown in the inset in the center right.

Figure 3

Current-voltage relationship of developing and tail currents in the absence and presence of 0.15 to 1.5 μg KCNE5-WT. A: Current-voltage relationship of peak developing current. B: Current-voltage relationship of tail current measured upon repolarization to -40 mV. C: Bar graphs showing current densities of peak developing current measured during test pulse to +60 mV. D: Bar graphs showing current densities of tail current measured upon repolarization to -40 mV following a test pulse to +60 mV. *P <.05; **P <.01 vs I_Ks.

Figure 4

Current-voltage relationship of developing and tail currents in the absence and presence of 1.5 μg KCNE5-WT or KCNE5-L65F. A: Representative current trace of I_Ks/CHO cells transfected with 1.5 μg WT KCNE5. B: Representative current trace of I_Ks/CHO cells transfected with 1.5 μg L65F KCNE5. Pulse protocol is shown in the inset in the center right. C: Current-voltage relationship of peak developing current. D: Current-voltage relationship of tail current measured upon repolarization to -40 mV. *P <.05 vs I_Ks+KCNE5-WT.

Figure 5

Effect of 0.3 and 1.5 μg KCNE5-WT or KCNE5-L65F on I_Ks. A: Bar graph showing density of peak developing current recorded during a test pulse to +60 mV. B: Bar graph showing density of tail currents recorded during repolarization to -40 mV following a test pulse to +60 mV. Statistical significance determined using analysis of variance. *P <.05; **P <.01.

Figure 6

Current-voltage relationship of developing and tail currents in cells cotransfected with equal amounts of KCNE5-WT and KCNE5-L65F. A: Representative current trace of I_Ks/CHO cells transfected with 0.75 μg WT KCNE5 and 0.75 μg L65F KCNE5. B: Representative current trace of I_Ks/CHO cells transfected with 1.5 μg WT KCNE5 and 1.5 μg L65F KCNE5. C: Current-voltage relationship of peak developing current. D: Current-voltage relationship of tail current measured upon repolarization to -40 mV. E: Bar graph showing density of peak developing current recorded during a test pulse to +60 mV (I_Ks; I_Ks+1.5 μg KCNE5-WT; I_Ks+0.75 μg KCNE5-WT+0.75 μg KCNE5-L65F; I_Ks+1.5 μg KCNE5-WT+1.5 μg KCNE5-L65F). F: Bar graph showing density of tail currents recorded during repolarization to -40 mV following a test pulse to +60 mV (I_Ks; I_Ks+1.5 μg KCNE5-WT; I_Ks+0.75 μg KCNE5-WT+0.75 μg KCNE5-L65F; I_Ks+1.5 μg KCNE5-WT+1.5 μg KCNE5-L65F).