2015 Volume 38 Issue 5 Pages 781-784
The human ether-à-go-go-related gene (hERG) channel mediates the rapid delayed rectifier potassium current (IKr) responsible for shaping the repolarization phase of cardiac action potentials. hERG mutation may cause hERG channel malfunction, leading to long QT syndrome and other arrhythmic disorders. Elucidation of the genotype–phenotype relationships of individual hERG mutations is key to the development of treatment for such arrhythmic disorders. We previously identified hERG(G487R), a missense mutant with a glycine-to-arginine substitution at position 487. In the absence of arrhythmogenic factors, hERG(G487R) subunit-containing channels show normal surface expression and gating kinetics. However, it remains unknown whether the mutation exacerbates hERG channel malfunction induced by arrhythmogenic factors. Here we used a voltage-clamp technique to compare the effects of the major arrythmogenic factors on wild-type hERG [hERG(WT)] and hERG(G487R) channel currents (IhERG) in HEK-293T cells. The extent of IhERG blockade by the antiarrhythmic drug dofetilide or E4031 was not different between these channels. On the other hand, the extracellular K+ concentration ([K+]ex)-dependent changes in the rates of recovery from inactivation and deactivation of IhERG were rather less obvious for hERG(G487R) channel than for hERG(WT) channel. These findings suggest that the inheritance of hERG(G487R) does not increase the risk of arrhythmic disorders induced by antiarrhythmic drugs or hypokalemia.
The human ether-à-go-go-related gene (hERG) encodes the alpha subunit of the rapid delayed rectifier potassium current (IKr)-mediating channel.1,2) hERG channel recovers from inactivation at the repolarization phase of an action potential and contributes to the prevention of premature action potential regeneration.1,2) hERG channel malfunction due to a genetic mutation may cause long QT syndrome and other arrhythmic disorders.1,2)
We previously indentified hERG(G487R) in a Japanese family with a member who died from sudden cardiac death.3) The family members carried hERG(G487R) and/or SCN5A(R1193Q), a voltage-gated Na+ channel gene variant.3) Although SCN5A(R1193Q) could underlie the symptom, its phenotypic penetrance is relatively low.4) Moreover, the glycine substitution could possibly cause hERG channel malfunction, affecting the subunit’s structural flexibility.5) In an attempt to assess the phathogenicity of hERG(G487R), we previously compared the hERG(G487R)-containing channels with wild-type hERG [hERG(WT)] channel and found that these channels are not different in surface expression and gating kinetics in the absence of arrhythmogenic factors.3)
To further confirm the non-pathogenicity of hERG(G487R), it is important to examine whether the G487R mutation increases the risk of arrhythnogenic factor-induced arrhythmic disorders. Several genetic variations directly or indirectly affecting hERG channel function are related to arrhythmic disorders induced by class III antiarrhythmic drugs or hypokalemia.6–8) We compared the effects of these arrhythmogenic factors on hERG(WT) and hERG(G487R) channel currents in HEK-293T cells. These results provide the scaffolding for designing treatment programs for hERG(G487R) carriers.
The details of the methods are given in the Supplementary Text. Briefly, HEK-293T cells were transfected with pCAG GS plasmid vector containing hERG(WT) or hERG(G487R) and that containing enhanced green fluorescent protein gene (WT and GR cells, respectively).
Two days later, ruptured-patch whole-cell recordings were made from green fluorescence-positive cells. The recording pipette contained (in mM) 134 potassium D-gluconic acid, 7.6 KCl, 9 KOH, 10 NaCl, 1.2 MgCl2, 10 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 0.5 ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and 4 ATP-Mg (pH 7.3). The bathing saline contained (in mM) 147 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose (pH 7.4, ca. 37°C). The holding potential was −80 mV. Saline containing a control or test agent was applied to the cell through a double-barrel tubing. Numerical data are expressed as mean±standard error of the mean (S.E.M.) throughout this article.
We monitored the peak amplitude of IhERG tail (Itail) periodically (interval, 7 s; Fig. 1A). A continuous application of dofetilide, a class III antiarrhythmic drug gradually reduced the Itail. The reduction reached the quasi-steady-state typically on the 5–25th trial after the application onset. The dose–response relation was similar between the WT and GR cells; Itail at the quasi-steady-sate was not different between the WT and GR cells for all the tested doses [p>0.05, Van der Waerden (VDW) test; Fig. 1B]. The dissociation constants and Hill coefficients estimated from the fitted Hill equations were 99.9 nM and 1.05 for the WT cells and 100.0 nM and 0.99 for the GR cells, respectively.
(A) Representative current responses to a double-step voltage stimulus (schematic) during an application of the control vehicle (basal) or the labeled dose of dofetilide. The traces of the dofetilide-affected responses were obtained after the blockade reached the quasi-steady-state. Each set of traces was obtained from a distinct cell. (B) Itail at the quasi-steady-state plotted against the dose of dofetilide (n, 3–12 WT cells and 4–11 GR cells). For each cell, the mean of the Itail measured on 3 trials before the dofetilide application was taken as 100%. p>0.05 (VDW test) between the WT and GR cells for all the tested doses. Sigmoid curves, Hill equations fitted to the data.
Moreover, at a concentration (30 nM) close to the apparent dissociation constant for hERG(WT) channel,8) E4031, an experimental antiarrhythmic drug reduced the Itail in the WT and GR cells by similar extents (31.5±2.5%, n=20 and 29.1±2.8%, n=14, respectively; p>0.05, VDW test; not illustrated).
We compared IhERG at a normal extracellular K+ concentration ([K+]ex) (5.4 mM) and a clinically observed hypokalemic [K+]ex (3.0 mM).7) Both hERG(WT) and hERG(G487R) channels appeared to mediate similar levels of whole-cell conductance at these [K+]ex when nearly fully activated (Supplementary Text). The WT or GR cells did not display a clear [K+]ex-dependent Itail increase9) (Supplementary Text).
In the WT cells (n=21), the time-constants of recovery from inactivation (1.88±0.10 ms at a [K+]ex of 3.0 mM and 2.21±0.16 ms at a [K+]ex of 5.4 mM; p<0.01, paired t-test; Fig. 2A, B) and deactivation (fast and slow components, 76.8±3.9 ms and 410.0±18.4 ms at a [K+]ex of 3.0 mM, 93.7±6.1 ms and 518.3±31.2 ms at a [K+]ex of 5.4 mM, respectively; p<0.01 for both the components, paired t-test; Fig. 2A, C, D) increased with the [K+]ex. In the GR cells (n=22), in contrast, the time-constants of recovery from inactivation (1.96±0.15 ms at a [K+]ex of 3.0 mM and 2.09±0.16 ms at a [K+]ex of 5.4 mM; p>0.05; Fig. 2A, B) and deactivation (fast and slow components, 85.9±5.5 ms and 468.3±30.9 ms at a [K+]ex of 3.0 mM, 79.8±5.5 ms and 478.6±35.3 at a [K+]ex of 5.4 mM, respectively; p>0.05 for both the components; Fig. 2A, C, D) did not increase with the [K+]ex. In both the cells, the relative amplitudes of the fast and slow components decreased and increased with the [K+]ex, respectively (Fig. 2E, F), indicating that extracellular K+ affects the fast and slow gating mechanisms in different manners.
(A) Representative IhERG tails elicited by a double-step voltage stimulus (schematic) during an application of saline containing 3.0 or 5.4 mM K+. Each set of traces was obtained from a distinct cell. White lines, single- and double-exponential functions fitted to the phases of IhERG reflecting recovery from inactivation (arrows) and deactivation (arrow heads), respectively. Superimposed traces, scaled close-ups of the phases of IhERG. (B–F) Time-constants of the fitted single-exponential functions (B) and time-constants and relative amplitudes of the fast and slow components of the fitted double-exponential functions (C–F). Raw data from the same cell are connected by a line. Mean data were collected from 21 WT cells and 22 GR cells. *, **, and ns, p<0.05, p<0.01, and p>0.05, respectively, paired t-test.
We found similar susceptibilities to the antiarrhythmic drug between hERG(G487R) and hERG(WT) channels (Fig. 1). This result suggests that clinical administration of class III antiarrhythmic drugs may not exert more harmful effects on hERG(G487R) carriers than on non-carriers.
It has been reported that a relatively large decrease in the [K+]ex attenuates hERG channel conductance as well as accelerates the recovery from inactivation and deactivation of hERG channel.9,10) The attenuation may compensate for a [K+]ex-dependent K+ driving force increase, whereas the acceleration disturbs the timing of IKr generation and thus may increase the risk of arrhythmic disorders. The relatively small [K+]ex shift mimicking normal and hypokalemic conditions used in this study did not produce an obvious Itail change (see Results) in both the WT and GR cells, indicating that the possible conductance change counterbalanced the K+ driving force change. On the other hand, the [K+]ex shift slightly changed the rates of recovery from inactivation and deactivation in the WT cells but not the GR cells (Fig. 2). These results suggest that the G487R mutation rather confers hERG channel a resistivity against the [K+]ex-dependent gating modulations and thus may not facilitate the induction of arrhythmic disorders under hypokalemia.
Our previous study showed that hERG(G487R)-containing channels are normal in surface expression, the time- and voltage-dependences of activation, deactivation, inactivation, and recovery from inactivation in the absence of the arrhythmogenic factors.3) The present and previous findings together suggest that hERG(G487R) may not increase the risk of arrhythmic disorders in the absence or presence of the arrhythmogenic factors. In the case of the subject family, SCN5A(R1193Q) might play a more decisive role in the development of the cardiac symptom than hERG(G487R).
This work was supported by grants from Toyama First Bank Foundation and University of Toyama’s presidential office.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.