Voltage-Dependent Gating of hERG Potassium Channels

The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor

Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome

The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (Kv) channels KCNQ1 and hERG as well as the voltage-gated sodium (Nav) channel encoded by SCN5A. Each channel performs a highly distinct function, despite sharing a common topology and structural components. These three channels are also the primary proteins mutated in congenital long QT syndrome (LQTS), a genetic condition that predisposes to cardiac arrhythmia and sudden cardiac death due to impaired repolarization of the action potential and has a particular proclivity for reentrant ventricular arrhythmias. Recent cryo-electron microscopy structures of human KCNQ1 and hERG, along with the rat homolog of SCN5A and other mammalian sodium channels, provide atomic-level insight into the structure and function of these proteins that advance our understanding of their distinct functions in the cardiac action potential, as well as the molecular basis of LQTS. In this review, the gating, regulation, LQTS mechanisms, and pharmacological properties of KCNQ1, hERG, and SCN5A are discussed in light of these recent structural findings

Modulation of hERG K+ Channel Deactivation by Voltage Sensor Relaxation

The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, Ikr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Slow deactivation is one of the hallmark features of the unusual gating characteristics of hERG channels, and plays a crucial role in providing a robust current that aids repolarization of the cardiac action potential. As such, there is significant interest in elucidating the underlying mechanistic determinants of slow hERG channel deactivation. Recent work has shown that the hERG channel S4 voltage sensor is stabilized following activation in a process termed relaxation. Voltage sensor relaxation results in energetic separation of the activation and deactivation pathways, producing a hysteresis, which modulates the kinetics of deactivation gating. Despite widespread observation of relaxation behaviour in other voltage-gated K+ channels, such as Shaker, Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase Ci-VSP, the relationship between stabilization of the activated voltage sensor by the open pore and voltage sensor relaxation in the control of deactivation has only recently begun to be explored. In this review, we discuss present knowledge and questions raised related to the voltage sensor relaxation mechanism in hERG channels and compare structure-function aspects of relaxation with those observed in related ion channels. We focus discussion, in particular, on the mechanism of coupling between voltage sensor relaxation and deactivation gating to highlight the insight that these studies provide into the control of hERG channel deactivation gating during their physiological functioning

Voltage-gating and assembly of split Kv10.1 channels

Voltage-gated ion channels allow ions to pass cell membrane upon changes of transmembrane electrical potential. Conformational changes in the voltage-sensing domain of the channel (VSD) are assumed to be transmitted to the pore domain (PD) through an alpha-helical linker between them (S4-S5 linker). We have previously shown that expression of VSD and PD as separate fragments results in functional Kv10.1 channels that retain voltage-dependence. Here we used such ‘split’ channels to investigate functional interactions between VSD and PD. We found that their electrophysiological properties greatly depend on where the S4-S5 linker is interrupted. Remarkably, wild-type-like channel behavior could be fully or largely restored by mutations of crucial linker amino acids, indicating that precise functional interactions between VSD and PD remain when they are not covalently bound. Voltage-Clamp Fluorometry measurements revealed that VSD motion is alerted in specific split channels, but these changes were subtler. Finally, the increased separation between VSD activation and channel opening in the split channel carrying a large deletion in the S4-S5 linker, as well as the failure of the PD expressed alone to give currents, suggest that the role of the VSD in the is to open the channel pore and prevent it from closing

Voltage-gating and assembly of split Kv10.1 channels

Voltage-gated ion channels allow ions to pass cell membrane upon changes of transmembrane electrical potential. Conformational changes in the voltage-sensing domain of the channel (VSD) are assumed to be transmitted to the pore domain (PD) through an alpha-helical linker between them (S4-S5 linker). We have previously shown that expression of VSD and PD as separate fragments results in functional Kv10.1 channels that retain voltage-dependence. Here we used such ‘split’ channels to investigate functional interactions between VSD and PD. We found that their electrophysiological properties greatly depend on where the S4-S5 linker is interrupted. Remarkably, wild-type-like channel behavior could be fully or largely restored by mutations of crucial linker amino acids, indicating that precise functional interactions between VSD and PD remain when they are not covalently bound. Voltage-Clamp Fluorometry measurements revealed that VSD motion is alerted in specific split channels, but these changes were subtler. Finally, the increased separation between VSD activation and channel opening in the split channel carrying a large deletion in the S4-S5 linker, as well as the failure of the PD expressed alone to give currents, suggest that the role of the VSD in the is to open the channel pore and prevent it from closing

Probing the molecular and structural basis of voltage sensor gating in Kv11.1 ion channels

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane, and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels but the role of this region of the channel is largely unexplored. In part this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro405 to Phe431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His402 and Tyr403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. We demonstrate that for Kv11.1, activation and deactivation processes are not simple reversal transitions, but rather deactivation and inactivation processes are likely to be coupled. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats

Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking

AbstractThe antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K+ external solution ([K+]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone’s inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A