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The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C.

ABSTRACT: hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I(Kr), which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37°C. Whole-cell measurements of current (I(hERG)) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V(0.5)) of L532P I(hERG) was positively shifted by ~+36mV compared to WT I(hERG); however at negative voltages a pronounced L532P I(hERG) was observed. Both activation and deactivation time-courses were accelerated for L532P I(hERG). The inactivation V(0.5) for L532P I(hERG) was shifted by ~+32mV. Under action potential (AP) voltage-clamp, L532P I(hERG) exhibited a dome-shaped current peaking at ~+16mV, compared to ~-31mV for WT-I(hERG). The L532P mutation produced an ~5-fold increase in the IC(50) for dronedarone inhibition of I(hERG). Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.

SUBMITTER: Zhang YH

PROVIDER: S-EPMC3245891 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C.

Zhang Yi H YH Colenso Charlotte K CK Sessions Richard B RB Dempsey Christopher E CE Hancox Jules C JC

Biochimica et biophysica acta 20110714 10

hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I(Kr), which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was exe ...[more]

PMID: 21777565

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Project description:Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

Project description:Congenital short QT syndrome (SQTS) is a repolarization disorder characterized by abbreviated QT intervals, atrial and ventricular arrhythmias and a risk of sudden death. This study characterized a missense mutation (I560T) in the S5 domain of the hERG K+ channel that has been associated with variant 1 of the SQTS. Whole cell patch clamp recordings of wild-type (WT) and I560T hERG current (IhERG) were made at 37 °C from hERG expressing HEK 293 cells, and the structural context of the mutation was investigated using a recently reported cryo-EM structure of hERG. Under conventional voltage clamp, the I560T mutation increased IhERG amplitude without altering the voltage-dependence of activation, although it accelerated activation time-course and also slowed deactivation time-course at some voltages. The voltage dependence of IhERG inactivation was positively shifted (by ∼24 mV) and the time-course of inactivation was slowed by the I560T mutation. There was also a modest decrease in K+ over Na+ ion selectivity with the I560T mutation. Under action potential (AP) voltage clamp, the net charge carried by hERG was significantly increased during ventricular, Purkinje fibre and atrial APs, with maximal IhERG also occurring earlier during the plateau phase of ventricular and Purkinje fibre APs. The I560T mutation exerted only a modest effect on quinidine sensitivity of IhERG: the IC50 for mutant IhERG was 2.3 fold that for WT IhERG under conventional voltage clamp. Under AP voltage clamp the inhibitory effect of 1 μM quinidine was largely retained for I560T hERG and the timing of peak I560T IhERG was altered towards that of the WT channel. In both the open channel structure and a closed hERG channel model based on the closely-related EAG structure, I560T side-chains were oriented towards membrane lipid and away from adjacent domains of the channel, contrasting with previous predictions based on homology modelling. In summary, the I560T mutation produces multiple effects on hERG channel operation that result in a gain-of-function that is expected to abbreviate ventricular, atrial and Purkinje fibre repolarization. Quinidine is likely to be of value in offsetting the increase in IhERG and altered IhERG timing during ventricular APs in SQTS with this mutation.

Project description:Ion channel behavior can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing, inactivating, and recovery) are commonly represented with Q10 coefficients or an Eyring relationship. In this article, we assess the validity of these representations by characterizing channel kinetics at multiple temperatures. We focus on the human Ether-à-go-go-Related Gene (hERG) channel, which is important in drug safety assessment and commonly screened at room temperature so that results require extrapolation to physiological temperature. In Part I of this study, we established a reliable method for high-throughput characterization of hERG1a (Kv11.1) kinetics, using a 15-second information-rich optimized protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using Chinese hamster ovary cells overexpressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, with temperature control. We characterize the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37°C and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients; it broadly follows a generalized, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol dependent. Our results show that a direct fit using our 15-s protocol best represents hERG kinetics at any given temperature and suggests that using the Generalized Eyring theory is preferable if no experimental data are available to derive model parameters at a given temperature.

Project description:Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1-354), including the eag domain (amino acids 1-135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (?2-135) or both the eag domain and the adjacent proximal domain (?2-354) had less rectifying current-voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1-135) or the eag domain plus the proximal domain (N1-354) with N-deleted hERG ?2-135 or hERG ?2-354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain-containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain-containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1-354 fragments nor the presence of the proximal domain in hERG ?2-135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG ?2-135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

Dataset Information

The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C.

Publications

The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C.

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