Functional effects of KCNE3 mutation and its role in the development of Brugada syndrome

Abstract

Introduction: The Brugada Syndrome (BrS), an inherited syndrome associated with a high incidence of sudden cardiac arrest, has been linked to mutations in four different genes leading to a loss of function in sodium and calcium channel activity. Although the transient outward current (I(to)) is thought to play a prominent role in the expression of the syndrome, mutations in I(to)-related genes have not been identified as yet.

Methods and results: One hundred and five probands with BrS were screened for ion channel gene mutations using single strand conformation polymorphism (SSCP) electrophoresis and direct sequencing. A missense mutation (R99H) in KCNE3 (MiRP2) was detected in one proband. The R99H mutation was found 4/4 phenotype positive and 0/3 phenotype-negative family members. Chinese hamster ovary (CHO)-K1 cells were co-transfected using wild-type (WT) or mutant KCNE3 and either WT KCND3 or KCNQ1. Whole-cell patch clamp studies were performed after 48 hours. Interactions between Kv4.3 and KCNE3 were analyzed in co-immunoprecipitation experiments in human atrial samples. Co-transfection of R99H-KCNE3 with KCNQ1 produced no alteration in current magnitude or kinetics. However, co-transfection of R99H KCNE3 with KCND3 resulted in a significant increase in the I(to) intensity compared to WT KCNE3+KCND3. Using tissues isolated from left atrial appendages of human hearts, we also demonstrate that K(v)4.3 and KCNE3 can be co-immunoprecipitated.

Conclusions: These results provide definitive evidence for a functional role of KCNE3 in the modulation of I(to) in the human heart and suggest that mutations in KCNE3 can underlie the development of BrS.

Keywords: Channelopathy; Electrophysiology; Genetics; Potassium Channels; Sudden Cardiac Death.

Figure 1

Pedigree of BrS family (A) and ECG recordings of proband and his brother (B-E). B: 12-lead ECG of 36 year old proband. C: ECG of proband recorded using telemetry leads during hospitalization showing initiation of VT. D: Initiation of VT recorded by ICD implanted in proband. E: 12-lead ECG of brother of proband.

Figure 2

Representative Kv7.1 currents recorded from CHO-K1 cells co-transfected with WT (A) or R99H (B) KCNE3. Membrane currents were elicited with voltage steps from a -80 mV holding potential to +60 mV. C-D. Mean current-voltage relationship for developing currents and tail currents for Kv7.1 channels co-expressed with WT- and R99H-KCNE3. In panel D continuous lines represent a Boltzmann function fit to the data. E-F. Time constant of activation and deactivation of Kv7.1 channels co-expressed with KCNE3 WT or R99H. Values in panels C-F are Mean±SEM of >8 experiments. * P<0.05 vs. Kv7.1+KCNE3 data.

Figure 3

Representative Kv4.3 currents recorded from CHO-K1 cells in the absence (A) and presence (B) of ancillary WT-KCNE3 subunits. Membrane currents were elicited by voltage steps from -80 mV (holding potential) to potentials between -50 and +50 mV. C: I-V relation for peak I_Kv4.3. I_Kv4.3 density is greater in the absence of WT KCNE3. Values shown represent Mean±SEM. “* P<0.05 vs Kv4.3+WT KCNE3.

Figure 4

Representative Kv4.3 currents recorded from CHO-K1 cells co-transfected with either WT (A) or R99H (B) KCNE3. The voltage protocol used is shown in the inset on top. C: I-V relation for peak I_Kv4.3. Average I_Kv4.3 density is greater in the presence of R99H KCNE3. D: Steady-state Kv4.3 inactivation curves in the absence and presence of WT- and R99H-KCNE3. Continuous lines represent a Boltzmann function fit to the data. Values shown represent Mean±SEM.

Figure 5

A: Inactivation time constants (τ) for I_Kv4.3 as a function of voltage. Values shown represent Mean±SEM. Inactivation time constants values were measured by fitting a monoexponential function to the current decay. *p<0.05 vs Kv4.3+WT KCNE3. #p<0.05 vs Kv4.3+WT KCNE3. B: Total charge of Kv4.3 during the first 40 ms as a function of voltage. *p<0.05 vs Kv4.3 alone. #p<0.05 vs Kv4.3+R99H KCNE3.

Figure 6

Representative traces Kv4.3+KCNE3 (A) and Kv4.3+R99H-KCNE3 currents (B) elicited using the protocol shown in the inset. I_Kv4.3 recovery was measured using twin voltage clamp steps to +50 mV from a holding potential of -80 mV separated by variable time intervals. (C) Ratio to peak current amplitude (P2) as a function of the interpulse interval. Values shown represent Mean±SEM.

Figure 7

A: I-V relation for peak I_Kv4.3 generated by channels formed by the co-expression of Kv4.3 plus WT-KCNE3, Kv4.3 plus R99H-KCNE3, or Kv4.3 plus WT-KCNE3 and R99H-KCNE3. B: Total charge of the current during the first 40 ms as a function of voltage. C: Steady-state Kv4.3 inactivation curves in the absence and presence of WT-, R99H-, or WT+R99H-KCNE3. Continuous lines represent a Boltzmann function fit to the data. Values shown represent Mean±SEM. * P<0.05 vs Kv4.3+WT KCNE3.

Figure 8

A: Co-immunoprecipitation of Kv4.3 channel complex with KCNE3 proteins isolated from human atrial tissues. The blots were probed with anti-Kv4.3 (upper) and anti-KCNE3 antibody (lower), respectively. IPP: immunoprecipitated proteins, SN: supernatant. B: Western blots showing Kv4.3 expression in untransfected CHO cells (lane 1), in rat ventricular myocardium (lane 2), and in rat ventricular myocardium when the sample was exposed to the antibody after incubating it with the antigenic peptide (lane 3) or when it was treated only with protein A-agarose (lane 4). C: Co-immunoprecipitation of Kv4.3 channel complex with KCNE3 proteins isolated from rat ventricular myocardium. The blots were probed with anti-Kv4.3 (upper) and anti-KCNE3 antibody (lower), respectively. Association of Kv4.3 and KCNE3 is produced in the absence of crosslinker (lanes 2 and 3), in the presence of DTBP (lane 4) or when a non-permeant crosslinker was used (BS, lane 5). No bands were observed in the case of untransfected CHO cells (lane 1).