HERG channel (dys)function revealed by dynamic action potential clamp technique

Abstract

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.

FIGURE 1

Diagram of the dAPC technique used to effectively replace the native I_Kr of a ventricular cell with I_HERG from a HEK-293 cell. (A) Overall experimental design. (B) Model-cell mode. I_HERG from a HEK-293 cell is recorded, scaled by a factor F_s, and then digitized (A/D) by a computer (PC), which contains a model of the human ventricular cell (Priebe and Beuckelmann, 1998), with I_Kr = 0. The momentary V_m is computed in real-time using the model equations and the inputted I_HERG. The computed V_m is converted into an analog signal (D/A), sent back to the amplifier, and applied as a voltage-clamp command to the HEK-293 cell. (C) Real-cell mode. The model cell has been replaced with a freshly isolated myocyte. I_HERG is recorded with amplifier 1, which is voltage-clamp mode, and scaled and applied as external current input (I_in) to amplifier 2, which is current clamp mode. The V_m of the myocyte (with I_Kr blocked pharmacologically), shaped by the input I_HERG, is applied as voltage-clamp command (V_cmd) to amplifier 1, thus establishing dAPC.

FIGURE 2

Characteristics of WT and R56Q I_HERG at 23 and 36°C. (A) Representative examples of WT (top), WT/R56Q (middle), and R56Q (bottom) currents elicited by a two-step voltage-clamp protocol. P1-activated I_HERG; steady-state current amplitude progressively increased and then decreased with depolarizing voltages, according to voltage-dependent inactivation. P2 elicited I_HERG tails; their peak is due to fast recovery from inactivation secondary to repolarization. The subsequent current decline is due to deactivation. (B) Voltage dependence of activation (protocol from A) and inactivation (protocol from inset). See Table 1, for half-maximal (in)activation voltage and slope factor values. (C) I-V relationships (peak of I_HERG tails during P2 plotted against voltage) of R56Q and WT channels.

FIGURE 3

Time constants of WT and R56Q I_HERG kinetics at 23 and 36°C. (A) Time constant of activation (τ_slow, triangles) and fast and slow time constant of deactivation (τ_fast and τ_slow, circles). Voltage-clamp protocols are shown in Fig. 2, A and C, respectively, and described in the Supplementary Material. Faster activation of R56Q HERG channels was apparent only at 36°C (see current traces inset), whereas deactivation was faster for R56Q than for WT at both 23 and 36°C (*, significant difference for R56Q versus WT, P < 0.05). WT/R56Q showed a mixed phenotype. (B) Time constants of inactivation (triangles) and recovery from inactivation (circles). Voltage-clamp protocols are shown as insets and described in the Supplementary Material.

FIGURE 4

The dAPC experiment with WT and R56Q I_HERG replacing I_Kr in the PB model cell. (A) WT I_HERG is an effective substitute for I_Kr. Superimposed APs (at 1 Hz) in the absence of I_Kr (long dashed line), with I_Kr (short dashed line), or with WT I_HERG (solid line, I_Kr = 0). (B) Time course of the AP waveform-elicited WT I_HERG is similar to that of I_Kr in the PB cell model except for the early activation (asterisk) phase. F_s for I_HERG was 0.008; see text for details. (C) APD₅₀ and APD₉₀ values at 1 and 2 Hz (*, significant difference for R56Q versus WT). (D) Representative APs with WT I_HERG (solid line) or R56Q I_HERG (shaded line), at 1 Hz (I_Kr = 0). (E and F) Boxed APs from D (E) and associated I_HERG (F) on an expanded timescale. The HERG currents were scaled to identical maximal amplitude values (F_s values indicated) and applied to the PB model cell as an external current input, and are thus responsible for repolarization of the model cell.

FIGURE 5

Regional AP heterogeneity is reproduced in a dAPC experiment. Subepicardial, M, and subendocardial APs were simulated at 1 Hz; note the different plateau levels and repolarization phases in these model cells (see the modified current densities in Table 2).

FIGURE 6

AP prolongation caused by the R56Q mutation in the three different cell types of Fig. 5. (A) Representative APs and (B) the corresponding I_HERG; note the increased inactivation of R56Q I_HERG (arrow) at the positive plateau-voltages of the subendocardial cell; (C) averaged APD₉₀ values at 1 and 2 Hz (*, significant difference for R56Q versus WT I_HERG).

FIGURE 7

AP characteristics of the subepicardial PB model cell. (A) Frequency dependence with I_Kr, WT I_HERG (n = 10), or R56Q I_HERG (n = 8) (*, significant difference for R56Q versus WT). (B) Phase-plane plot for the net membrane current (I_total) and I_HERG during repolarization (starting from ∼+18 mV during phase-1 repolarization). APs from which these phase planes were obtained were generated at 1 Hz and are shown in Fig. 4, D and E. Arrows indicate progression of time.

FIGURE 8

The dAPC experiment with I_HERG replacing I_Kr in rabbit myocytes. (A) Block of I_Kr with 5 μmol/L E-4031 (inset, pulse protocol). Superimposed tracings of typical recordings in absence (control) and presence of E-4031, and difference (E-4031 sensitive) current. Mean I_Kr density, determined from the E-4031 sensitive current, was 0.63 ± 0.1 pA/pF (n = 9). (B) APs in a myocyte stimulated at 0.2 Hz before and after applying E-4031. Superfusion of cells with E-4031 caused early after-depolarizations. (C and D) APs from a myocyte and associated WT (C) or R56Q I_HERG (D) at different frequencies. The myocyte was successively coupled to HEK-293 cells transfected with WT or R56Q HERG channels. Note the different I_HERG waveforms (*, transient I_HERG; arrow, sustained I_HERG) and frequency-dependent AP prolongation with R56Q (see also Table 2 in the Supplementary Material).

FIGURE 9

Action potential characteristics of rabbit ventricular myocytes with WT and R56Q I_HERG. (A) Superimposed APs from a single myocyte successively coupled to HEK-293 cells expressing WT (solid line) or R56Q I_HERG (shaded line), and the corresponding I_HERG waveforms at 1 and 4 Hz. (B–D) Frequency dependence of APD₉₀ prolongation (B; see also Table 2 in the Supplementary Material) and transient (C) and sustained (D) I_HERG amplitudes, each normalized to their values at 1 Hz. Asterisks indicate significant difference for R56Q versus WT.