Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines.
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ABSTRACT:
SUBMITTER: Guo J
PROVIDER: S-EPMC2735921 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Clozapine, a commonly used antipsychotic drug, can induce QT prolongation, which may lead to torsades de pointes and sudden death. To investigate the arrhythmogenic side effects of clozapine, we studied the impact of clozapine on human ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes and HEK293 cells, and on the delayed rectifier K(+) currents of guinea-pig cardiomyocytes. Clozapine dose-dependently decreased the amplitudes of the currents at the end of voltage steps, and the tail currents of HERG. The IC(50) for the clozapine blockade of HERG currents in Xenopus oocytes progressively decreased relative to depolarization (39.9 microM at -40 mV, 28.3 microM at 0 mV and 22.9 microM at +40 mV), whereas the IC(50) for the clozapine-induced blockade of HERG currents in HEK293 cells at 36 degrees C was 2.5 microM at +20 mV. The clozapine-induced blockade of HERG currents was time dependent: the fractional current was 0.903 of the control at the beginning of the pulse, but declined to 0.412 after 4 s at a test potential of 0 mV. The clozapine-induced blockade of HERG currents was use-dependent, exhibiting more rapid onset and greater steady state blockade at higher frequencies of activation, with a partial relief of blockade observed when the frequency of activation was decreased. In guinea-pig ventricular myocytes held at 36 degrees C, treatment with 1 and 5 microM clozapine blocked the rapidly activating delayed rectifier K(+) current (I(Kr)) by 24.7 and 79.6%, respectively, but did not significantly block the slowly activating delayed rectifier K(+) current (I(Ks)). Our findings collectively suggest that blockade of HERG currents and I(Kr), but not I(Ks), may contribute to the arrhythmogenic side effects of clozapine.
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Project description:Ultrasound strain imaging using 2-D speckle tracking has been proposed to quantitatively assess changes in myocardial contractility caused by ischemia. Its performance must be demonstrated in a controlled model system as a step toward routine clinical application. In this study, a well-controlled 2-D cardiac elasticity imaging technique was developed using two coplanar and orthogonal linear probes simultaneously imaging an isolated retroperfused rabbit heart. Acute ischemia was generated by left anterior descending (LAD) artery ligation. An excitation-contraction decoupler, 2,3-butanedione monoxime, was applied at a 4-mM concentration to reversibly reduce myocardial contractility. Results using a single probe demonstrate that directional changes in the in-plane principal deformation axes can help locate the bulging area as a result of LAD ligation, which matched well with corresponding Evans Blue staining, and strains or strain magnitude, based on principal stretches, can characterize heart muscle contractility. These two findings using asymmetric displacement accuracy (i.e., normal single-probe measurements with good axial but poor lateral estimates) were further validated using symmetric displacement accuracy (i.e., dual-probe measurements using only accurate axial tracking estimates from each). However, the accuracy of 2-D cardiac strain imaging using a single probe depends on the probe's orientation because of the large variance in lateral displacement estimates.
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Project description:BackgroundThe normalization of DNA microarrays allows comparison among samples by adjusting for individual hybridization intensities. The approaches most commonly used are global normalization methods that are based on the expression of all genes on the slide and on the modulation of a small proportion of genes. Alternative approaches must be developed for microarrays where the proportion of modulated genes and their distribution are unknown and they may be biased towards up- or down-modulated trends.ResultsThe aim of the work is to study the use of spike-in controls to normalize low-density microarrays. Our test-array was designed to analyze gene modulation in response to hypoxia (a condition of low oxygen tension) in a macrophage cell line. RNA was extracted from controls and cells exposed to hypoxia, mixed with spike RNA, labeled and hybridized to our test-array. We used eight bacterial RNAs as source of spikes. The test-array contained the oligonucleotides specific for 178 mouse genes and those specific for the eight spikes. We assessed the quality of the spike signals, the reproducibility of the results and, in general, the nature of the variability. The small values of the coefficients of variation revealed high reproducibility of our platform either in replicated spots or in technical replicates. We demonstrated that the spike-in system was suitable for normalizing our platform and determining the threshold for discriminating the hypoxia modulated genes. We assessed the application of the spike-in normalization method to microarrays in which the distribution of the expression values was symmetric or asymmetric. We found that this system is accurate, reproducible and comparable to other normalization methods when the distribution of the expression values is symmetric. In contrast, we found that the use of the spike-in normalization method is superior and necessary when the distribution of the gene expression is asymmetric and biased towards up-regulated genes.ConclusionWe demonstrate that spike-in controls based normalization is a reliable and reproducible method that has the major advantage to be applicable also to biased platform where the distribution of the up- and down-regulated genes is asymmetric as it may occur in diagnostic chips.
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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.
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Project description:Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K(+) channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K(+) channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.
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Project description:Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Long-QT syndrome type-2 (LQT2) and increased risk of cardiac arrhythmia. Reduced PM-expression is primarily attributed to retention and degradation of misfolded channels by endoplasmic reticulum (ER) protein quality control (QC) systems. However, as the molecular pathogenesis of LQT2 was defined using severely-misfolded hERG variants with limited PM-expression, the potential contribution of post-ER (peripheral) QC pathways to the disease phenotype remains poorly established. Here, we investigate the cellular processing of mildly-misfolded Per-Arnt-Sim (PAS)-domain mutant hERGs, which display incomplete ER-retention and PM-expression defects at physiological temperature. We show that the attenuated PM-expression of hERG is dictated by mutation-specific contributions from both the ER and peripheral QC systems. At the ER, PAS-mutants experience inefficient conformational maturation coupled with rapid ubiquitin-dependent proteasomal degradation. In post-ER compartments, they are rapidly endocytosed from the PM via a ubiquitin-independent mechanism and rapidly targeted for lysosomal degradation. Conformational destabilization underlies aberrant cellular processing at both ER- and post-ER compartments, since conformational correction by a hERG-specific pharmacochaperone or low-temperatures can restore WT-like trafficking. Our results demonstrate that the post-ER QC alone or jointly with the ER QC determines the loss-of-PM-expression phenotype of a subset of LQT2 mutations.
| S-EPMC6465299 | 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.
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Project description:Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K(+) current (IKr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.
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Project description:In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.
| S-EPMC3585479 | 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