Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome

Abstract

The K⁺ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K⁺ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy.

Keywords: arrhythmia; cardiac pathophysiology; channelopathy; electrophysiology; long QT syndrome.

Figure 1

A, Pedigree of the family carrying the G1006fs/49 mutant of the KCNH2 channel. Filled symbols indicate clinically affected individuals; + indicates positive for the mutation; arrow indicates the index patient. B, The index patient and (C) her brother had prolonged QT intervals corrected for heart rate (QTc) on electrocardiogram. D, The index patient was treated with beta‐blocker therapy and received an implantable cardioverter defibrillator (ICD) but continued to exhibit tachyarrhythmic episodes that were recorded on ICD interrogation

Figure 2

A, Confocal analysis of HEK293 cell expressing either GFP‐tagged KCNH2 homotetramers (KCNH2) or mCherry‐tagged G1006fs/49 homotetramers (G1006fs/49). B, Western blotting analysis using cell lysate of HEK293 cells expressing either GPP‐tagged KCNH2 homotetramers (KCNH2) or mCherry‐tagged G1006fs/49 homotetramers (G1006fs/49). The bands were detected using the antibodies against the fluorescent tags. C, Densitometric analysis performed on 3 independent Western blotting experiments. D, Confocal analysis of HEK293 cells expressing simultaneously GFP‐tagged KCNH2 channels (KCNH2) and mCherry‐tagged G1006fs/49 channels (G1006fs/49)

Figure 3

K⁺ currents recorded for (A) KCNH2 homotetramers, (B) G1006fs/49 homotetramers and (C) KCNH2‐G1006fs/49 heterotetramers in HEK293 cells. D, I‐V plot of the K⁺ currents measured at the end of the depolarizing steps for KCNH2 homotetramers (green trace, cells = 10), G1006fs/49 homotetramers (red trace, cells = 9) and KCNH2‐G1006fs/49 heterotetramers (orange trace, cells = 10). E, Activation curves measured with K⁺ normalized tail currents fitted to a Boltzmann relationship. ANOVA test, P ≤ 0.05; *KCNH2 vs KCNH2‐G1006fs/49; ^#KCNH2 vs G1006fs/49. F, Representation of V _1/2 and κ values. ANOVA test, P ≤ 0.05; *KCNH2 vs KCNH2‐G1006fs/49; ^#KCNH2 vs G1006fs/49

Figure 4

Voltage clamp protocol used to analyse channel inactivation and relative traces recorded for (A) KCNH2 homotetramers, (B) G1006fs/49 homotetramers and (C) KCNH2‐G1006fs/49 heterotetramers in HEK293 cells. D, Representation of the time constant of inactivation (tau) vs membrane potential obtained by fitting the current with a single‐exponential function for KCNH2 homotetramers (green trace, cells = 4), G1006fs/49 homotetramers (red trace, cells = 4) and KCNH2‐G1006fs/49 heterotetramers (orange trace, cells = 4). ANOVA test, P ≤ 0.05; *KCNH2 vs KCNH2‐G1006fs/49; ^#KCNH2 vs G1006fs/49

Figure 5

Voltage clamp protocol used to analyse channel recovery from inactivation and 100 ms of a relative trace recorded for (A) KCNH2 homotetramers, (B) G1006fs/49 homotetramers and (C) KCNH2‐G1006fs/49 heterotetramers in HEK293 cells. D, Time constant of inactivation over the membrane potential of KCNH2 homotetramers (green trace, cells = 7), G1006fs/49 homotetramers (red trace, cells = 6) and KCNH2‐G1006fs/49 heterotetramers (orange trace, cells = 8). ANOVA test, P ≤ 0.05; ^#KCNH2 vs G1006fs/49; *KCNH2 vs KCNH2‐G1006fs/49

Figure 6

Representative traces of the KCNH2 tail currents with and without NS1643 (A), representative traces of KCNH2 currents used for evaluating the channel inactivation rate with and without NS1643 (B), representative traces of KCNH2 currents used for evaluating the rate of recovery from inactivation with and without NS1643 (C). The activation curve, (D) the time constant of inactivation (E) and recovery from inactivation (F) for KCNH2 homotetramers either in control extracellular solution (green trace, cells = 6) or after administration by perfusion of NS1643 30 µmol/L (black traces, cells = 6). ANOVA test, P ≤ 0.05, *KCNH2 vs KCNH2 5 min NS1643, ^XKCNH2 vs KCNH2 10 min NS1643

Figure 7

Representative traces of the G1006fs/49 (A) and KCNH2‐G1006fs/49 (B) tail currents with and without NS1643 used for evaluating the channel activation kinetics. Effect of 30 µmol/L NS1643 on (C) G1006fs/49 homotetramers and (D) KCNH2‐G1006fs/49 heterotetramers activation kinetics. The activation curve is significantly left‐shifted by 30 µmol/L NS1643 for both G1006fs/49 homotetramers (C, red trace, cells = 5) and KCNH2‐G1006fs/49 heterotetramers (D, orange trace, cells = 5). In both C and D plots, the green trace is the activation curve for KCNH2 homotetramers. ANOVA test, P ≤ 0.05 *G1006fs/49 vs G1006fs/49 NS1643 in C; P ≤ 0.05 *KCNH2‐G1006fs/49 vs KCNH2‐G1006fs/49 NS1643 in D