HERG potassium channel regulation by the N-terminal eag domain.
Ontology highlight
ABSTRACT:
SUBMITTER: Gustina AS
PROVIDER: S-EPMC4793660 | biostudies-literature | 2012 Aug
REPOSITORIES: biostudies-literature
Publications
HERG potassium channel regulation by the N-terminal eag domain.
Gustina Ahleah S AS Trudeau Matthew C MC
Cellular signalling 20120413 8
Human ether-á-go-go related gene (hERG, K(v)11.1) potassium channels play a significant role in cardiac excitability. Like other K(v) channels, hERG is activated by membrane voltage; however, distinct from other K(v) channels, hERG channels have unusually slow kinetics of closing (deactivation). The mechanism for slow deactivation involves an N-terminal "eag domain" which comprises a PAS (Per-Arnt-Sim) domain and a short Cap domain. Here we review recent advances in understanding how the eag dom ...[more]
Similar Datasets
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.
| S-EPMC3557309 | biostudies-literature
Project description:Human ether-à-go-go-related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an "intrinsic ligand" that is composed of residues 860-862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS-CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG "core" channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.
| S-EPMC6445578 | biostudies-literature
Project description:The KCNH voltage-dependent potassium channels (ether-à-go-go, EAG; EAG-related gene, ERG; EAG-like channels, ELK) are important regulators of cellular excitability and have key roles in diseases such as cardiac long QT syndrome type 2 (LQT2), epilepsy, schizophrenia and cancer. The intracellular domains of KCNH channels are structurally distinct from other voltage-gated channels. The amino-terminal region contains an eag domain, which is composed of a Per-Arnt-Sim (PAS) domain and a PAS-cap domain, whereas the carboxy-terminal region contains a cyclic nucleotide-binding homology domain (CNBHD), which is connected to the pore through a C-linker domain. Many disease-causing mutations localize to these specialized intracellular domains, which underlie the unique gating and regulation of KCNH channels. It has been suggested that the eag domain may regulate the channel by interacting with either the S4-S5 linker or the CNBHD. Here we present a 2 Å resolution crystal structure of the eag domain-CNBHD complex of the mouse EAG1 (also known as KCNH1) channel. It displays extensive interactions between the eag domain and the CNBHD, indicating that the regulatory mechanism of the eag domain primarily involves the CNBHD. Notably, the structure reveals that a number of LQT2 mutations at homologous positions in human ERG, in addition to cancer-associated mutations in EAG channels, localize to the eag domain-CNBHD interface. Furthermore, mutations at the interface produced marked effects on channel gating, demonstrating the important physiological role of the eag domain-CNBHD interaction. Our structure of the eag domain-CNBHD complex of mouse EAG1 provides unique insights into the physiological and pathophysiological mechanisms of KCNH channels.
| S-EPMC3910112 | biostudies-literature
Project description:The inward rectifier voltage-gated potassium channel hERG is of primary importance for the regulation of the membrane potential of cardiomyocytes. Unlike most voltage-gated K(+)-channels, hERG shows a low elementary conductance at physiological voltage and potassium concentration. To investigate the molecular features underlying this unusual behavior, we simulated the ion conduction through the selectivity filter at a fully atomistic level by means of molecular dynamics-based methods, using a homology-derived model. According to our calculations, permeation of potassium ions can occur along two pathways, one involving site vacancies inside the filter (showing an energy barrier of about 6 kcal mol(-1)), and the other characterized by the presence of a knock-on intermediate (about 8 kcal mol(-1)). These barriers are indeed in accordance with a low conductance behavior, and can be explained in terms of a series of distinctive structural features displayed by the hERG ion permeation pathway.
| S-EPMC3487835 | biostudies-literature
Project description:The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.
| S-EPMC3398701 | biostudies-literature
Project description:Based on the scaffold of astemizole and E-4031, four AIE light-up probes (L1-L4) for Human Ether-a-go-go-Related Gene (hERG) potassium channel were developed herein using AIE fluorogen(TPE). These probes showing advantages such as low background interference, superior photostability, acceptable cell toxicity, and potent inhibitory activity, which could be used to image hERG channels at the nanomolar level. These AIE light-up probes hoped to provide guidelines for the design of more advanced AIE sensing and imaging hERG channels to a broad range of applications.
| S-EPMC6375833 | biostudies-literature
Project description:Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.
| S-EPMC2823420 | biostudies-literature
Project description:BackgroundIvabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN-channels. This study investigated the propensity of ivabradine to interact with KCNH2-encoded human Ether-à-go-go-Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia.Methods and resultsPatch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Ih ERG was inhibited with an IC50 of 2.07 μmol/L for the hERG 1a isoform and 3.31 μmol/L for coexpressed hERG 1a/1b. The voltage and time-dependent characteristics of Ih ERG block were consistent with preferential gated-state-dependent channel block. Inhibition was partially attenuated by the N588K inactivation-mutant and the S624A pore-helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK-based homology model of hERG, the 2 aromatic rings of the drug could form multiple π-π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea-pig Langendorff-perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of the MAPD90 restitution curve.ConclusionsIvabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits Ih ERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design.
| S-EPMC4579960 | biostudies-literature
Project description:Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.
| S-EPMC3057830 | biostudies-literature
Project description:The members of the family of voltage-gated KCNH potassium channels play important roles in cardiac and neuronal repolarization, tumour proliferation and hormone secretion. These channels have a C-terminal cytoplasmic domain which is homologous to cyclic nucleotide-binding domains (CNB-homology domains), but it has been demonstrated that channel function is not affected by cyclic nucleotides and that the domain does not bind nucleotides in vitro. Here, the crystallization and preliminary crystallographic analysis of a CNB-homology domain from a member of the KCNH family, the mouse EAG channel, is reported. X-ray diffraction data were collected to 2.2 Å resolution and the crystal belonged to the hexagonal space group P3(1)21.
| S-EPMC3310546 | biostudies-literature