KCNE2 modulation of Kv4.3 current and its potential role in fatal rhythm disorders

Abstract

Background: The transient outward current I(to) is of critical importance in regulating myocardial electrical properties during the very early phase of the action potential. The auxiliary beta subunit KCNE2 recently was shown to modulate I(to).

Objective: The purpose of this study was to examine the contributions of KCNE2 and its two published variants (M54T, I57T) to I(to).

Methods: The functional interaction between Kv4.3 (alpha subunit of human I(to)) and wild-type (WT), M54T, and I57T KCNE2, expressed in a heterologous cell line, was studied using patch-clamp techniques.

Results: Compared to expression of Kv4.3 alone, co-expression of WT KCNE2 significantly reduced peak current density, slowed the rate of inactivation, and caused a positive shift of voltage dependence of steady-state inactivation curve. These modifications rendered Kv4.3 channels more similar to native cardiac I(to). Both M54T and I57T variants significantly increased I(to) current density and slowed the inactivation rate compared with WT KCNE2. Moreover, both variants accelerated the recovery from inactivation.

Conclusion: The study results suggest that KCNE2 plays a critical role in the normal function of the native I(to) channel complex in human heart and that M54T and I57T variants lead to a gain of function of I(to), which may contribute to generating potential arrhythmogeneity and pathogenesis for inherited fatal rhythm disorders.

Figure 1

KCNE2 co-expression with Kv4.3 produces smaller I_to-like currents with slower activation/inactivation kinetics. A: Representative current traces recorded from Chinese hamster ovary (CHO) cells expressing Kv4.3 (left) and Kv4.3 + KCNE2 (right). As shown in the inset in panel A, depolarizing step pulses of 1-second duration were introduced from a holding potential of −80 mV to potentials ranging from −40 to +50 mV in 10-mV increments. B: Current–voltage relationship curve showing peak current densities in the absence and presence of co-transfected KCNE2 (*P <.05 vs Kv4.3). C: Bar graphs showing the kinetic properties of reconstituted channel currents: time to peak of activation course (left) and inactivation time constants (right) measured using test potential to +20 mV (*P <.05 vs Kv4.3). Numbers in parentheses indicate numbers of experiments. D: Normalized conductance–voltage relationship for peak outward current of Kv4.3 and Kv4.3 + KCNE2 channels.

Figure 2

KCNE2 co-expression with Kv4.3 causes a positive shift of voltage dependence of steady-state inactivation. A: Representative Kv4.3 and Kv4.3 + KCNE2 current traces induced by 500-ms pulses (P1) from −90 to +50 mV applied from the holding potential −80 mV in 10-mV steps followed by a second pulse (P2) to +40 mV. B: Steady-state inactivation curves for Kv4.3 (open circles) and Kv4.3 + KCNE2 (closed circles) channels.

Figure 3

Effects of KCNE2 co-expression on recovery from inactivation of Kv4.3 (A) and Kv4.3 + KChIP2b (B) currents. Recovery from inactivation was assessed by a two-pulse protocol (A, inset): a 400-ms test pulse to +50 mV (P1) followed by a variable interval at −80 mV, then by a second test pulse to +50 mV (P2). Data were fit to a single exponential.

Figure 4

Two KCNE2 transmembrane variants, I57T and M54T, increase the reconstituted Kv4.3 + KChIP2b channel current and slow its inactivation. A: Three sets of current traces elicited by depolarizing pulses for 500 ms from a holding potential of −80 mV to potentials ranging between −40 and +50 mV in 10-mV increments (same protocol as in experiments of Figure 1A). B: Superimposition of three original current traces recorded upon depolarization showing variant-related increase in peak outward current density. C: Current–voltage relationship curve showing average peak outward current densities (*P <.05 vs Kv4.3 + KChIP2b + WT). WT = wild type.

Figure 5

Two KCNE2 variants slow inactivation kinetics and accelerate recovery from inactivation. A, a: Three current traces obtained from Chinese hamster ovary (CHO) cells transfected with wild-type (WT), I57T, and M54T KCNE2 variant co-expressed with Kv4.3 and KChIP2b. Traces, which are normalized and superimposed, show that the variants slow inactivation. A, b: Time constants of decay at +20 mV for WT and variant KCNE2 (*P <.05 vs Kv4.3 + KChIP2b + WT). Numbers in parentheses indicate numbers of observations. B: Time constants of recovery from inactivation recorded using a double-pulse protocol (*P <.05 vs Kv4.3 + KChIP2b + WT). Numbers in parentheses indicate numbers of observations.