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. 2003 Sep 2;100(18):10534-9.
doi: 10.1073/pnas.1832721100. Epub 2003 Aug 19.

Gating currents associated with intramembrane charge displacement in HERG potassium channels

David R Piper  1 , Anthony Varghese, Michael C Sanguinetti, Martin Tristani-Firouzi
Affiliations

Gating currents associated with intramembrane charge displacement in HERG potassium channels

David R Piper et al. Proc Natl Acad Sci U S A. .

Abstract

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K+ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

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Figures

Fig. 1.
Fig. 1.
Comparison of gating and ionic currents for ether-a-go-go (EAG) and HERG K+ channels. EAG (a) and HERG (b) ionic current elicited by 300-ms depolarizations ranging from -50 to +30 mV, applied in 20-mV increments from a HP of -110 mV. Inset illustrates that a fast gating component of gating current precedes the onset of ionic current activation for the pulse to +30 mV. (c) I-V relationship for EAG (black triangles) and HERG (red squares). Current amplitude at the end of a 300-ms depolarization was normalized to the amplitude at 0 mV and the normalized values plotted versus test potential. Shown are EAG (d) and HERG (e) gating currents elicited by 80-ms (EAG) or 300-ms (HERG) depolarizations to +10 mV from a HP of -110 mV. Normalized and superimposed traces of IgON at +10 mV (f) and IgOFF at -110 mV (g) for EAG (black) and HERG (red) are compared on the same time scale.
Fig. 2.
Fig. 2.
Properties of WT HERG gating currents. Family of HERG gating currents elicited by 300-ms pulses to the indicated test potential from HPs of -110 mV (a) and 0 mV (b). Test pulses were applied in 10-mV increments. (c) Normalized QOFF-V and G-V relationships of WT HERG channels. The integral of IgOFF was normalized for each cell and the mean value plotted versus voltage. The isochronal G-V relationship was determined by tail current analysis of ionic currents elicited with 300-ms pulses. The voltage dependence of QOFF-V and G-V relationships was determined by fitting data to a Boltzmann function. The V1/2 and z were -25 ± 1.2 mV and 1.9 ± 0.06 for the QOFF-V relationship at a HP of -110 mV (n = 19), -5.6 ± 1.2 mV and 2.4 ± 0.1 for the G-V relationship (n = 9), and -83 ± 2.2 mV and 2.4 ± 0.05 for the QOFF-V relationship at a HP of 0 mV (n = 7). (d) Relative proportion of fast QON as a function of test potential. IgON was integrated, and the charge moved in the first 2 ms (Q2ms) was expressed as fraction of total charge moved (Q300ms) at each test potential (n = 7). (e) Voltage dependence of QFAST. Fast IgON was elicited by 2-ms pulses to potentials ranging from -110 to +150 mV from a HP of -120 mV. IgON was integrated after subtracting standing current at the end of the 2-ms pulse, plotted versus voltage and fitted to a Boltzmann function to determine relative QFAST. The V1/2 and z were +28 ± 4.4 mV and 0.7 ± 0.02 (n = 5).
Fig. 3.
Fig. 3.
Gating currents of inactivationdeficient S631A HERG channels. Family of gating currents elicited as in Fig. 2 from a HP of -110 mV (a) and 0 mV (b). Test pulses were applied in 20-mV increments. (c) Normalized QOFF-V and G-V relationships. The V1/2 and z were -21 ± 1.4 mV and 1.9 ± 0.1 for the QOFF-V relationship at a HP of -110 mV (n = 9), +9.4 ± 1.4 mV and 2.4 ± 0.1 for the G-V relationship (n = 7), and -82 ± 2.2 mV and 2.1 ± 0.1 for the QOFF-V relationship at a HP of 0 mV (n = 7). (d)Q2ms/Q300ms as a function of test potential (n = 4). (e) Voltage dependence of QFAST was determined as in Fig. 2e. The V1/2 and z were +32 ± 1.5 mV and 0.7 ± 0.01 (n = 5). (f and g) IgOFF for WT (f) and S631A (g) HERG elicited by pulses to -110 mV after a 300-ms test pulse to +30 mV (trace 1), 0 mV (trace 2), -30 mV (trace 3), and -60 mV (trace 4).
Fig. 4.
Fig. 4.
Comparison of WT and S631A channel inactivation. (a) Voltage dependence of ionic current inactivation. Peak current during the test pulse of the triple pulse protocol was plotted as a function of interpulse potential. The V1/2 and z of the Boltzmann function were -82 ± 2.4 mV and 1.1 ± 0.07 for WT (n = 3) and +7.8 ± 5.0 mV and 1.4 ± 0.06 for S631A (n = 3). (b) Gating currents for WT and S631A HERG elicited by a triple-pulse voltage protocol (prepulse = +40 mV, interpulse = -110 mV, test pulse = +40 mV). (c) Expanded time scale of boxed area in b for WT (Left) and S631A (Right) for interpulse potentials ranging from -120 to -40 mV in 20-mV increments. (d) Charge measured during test pulse of a triple pulse protocol divided by the maximal QOFF elicited by a 300-ms-step depolarization (QTP/Qmax) plotted versus interpulse test potential. Because external TEA is known to slow the recovery from and onset of HERG inactivation (3), TEA in the external solution was substituted with 120 mM NMG for experiments shown in b-d.
Fig. 5.
Fig. 5.
Markov model of WT and S631A HERG channels. (a) Schematic of the Markov state model. The rates and valences for the transitions are presented in Table 1. The forward rates of the transitions marked as red arrows were changed to model S631A behavior as indicated in red text. (b) Ionic currents recorded from WT HERG channels (Left) and currents predicted by the model (Right). Voltages in b-e are indicated by the following colors: black, +30 mV; red, +10 mV; green, -10 mV; blue, -30 mV; cyan, -50 mV. (c) Ionic currents recorded from S631A HERG channels (Left) and currents predicted by the model (Right). (d) Gating currents recorded from WT HERG channels (Left) and predicted by the model (Right). (e) Gating currents recorded from S631A HERG channels (Left) and Ig predicted by the model (Right). (f) WT HERG QOFF-V (HP = -110 mV, V1/2 = -23 mV, and z = 1.8; HP = 0 mV, V1/2 = -86 mV, and z = 2.9) and G-V curves (V1/2 = -9 mV and z = 2.2) predicted by the model. (g) S631A QOFF-V (HP = -110 mV, V1/2 = -20 mV, and z = 1.4; HP = 0 mV, V1/2 = -79 mV, and z = 2.4) and G-V curves (V1/2 = -2mV and z = 1.8) predicted by the model. (h)(Upper) Relative inactivation predicted by the model for a triple pulse protocol as in Fig. 4a. V1/2 and z were -88 mV and 1.0 for WT and +3 mV and 0.9 for S631A. (Lower) QTP/Qmax predicted by the model, as calculated in Fig. 4d.
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