NBD-Based Environment-Sensitive Fluorescent Probes for the Human Ether-a-Go-Go-Related Gene Potassium Channel.
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ABSTRACT:
SUBMITTER: Li Q
PROVIDER: S-EPMC8160426 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity.
| S-EPMC4741275 | biostudies-literature
Project description:The first small-molecule fluorogenic probe A1 for imaging the human Ether-a-go-go-Related Gene (hERG) potassium channel based on the photoinduced electron transfer (PET) off-on mechanism was described herein. After careful biological evaluation, this probe had the potential of detecting and imaging the hERG channel at the molecular and cellular level. Moreover, the competitive binding mechanism of this probe would presumably minimize the effects on the electrophysiological properties of the hERG channel. Therefore, this probe may serve as a powerful toolkit to the hERG-associated study.
| S-EPMC4374661 | biostudies-literature
Project description:BackgroundThe expression of the human Eag1 potassium channel (Kv10.1) is normally restricted to the adult brain, but it has been detected in both tumour cell lines and primary tumours. Our purpose was to determine the frequency of expression of Eag1 in soft tissue sarcoma and its potential clinical implications.ResultsWe used specific monoclonal antibodies to determine the expression levels of Eag1 in soft tissue sarcomas from 210 patients by immunohistochemistry. Eag1 was expressed in 71% of all tumours, with frequencies ranging from 56% (liposarcoma) to 82% (rhabdomyosarcoma). We detected differences in expression levels depending on the histological type, but no association was seen between expression of this protein and sex, age, grade or tumour size. Four cell lines derived from relevant sarcoma histological types (fibrosarcoma and rhabdomyosarcoma) were tested for Eag1 expression by real-time RT-PCR. We found all four lines to be positive for Eag1. In these cell lines, blockage of Eag1 by RNA interference led to a decrease in proliferation.ConclusionEag1 is aberrantly expressed in over 70% sarcomas. In sarcoma cell lines, inhibition of Eag1 expression and/or function leads to reduced proliferation. The high frequency of expression of Eag1 in primary tumours and the restriction of normal expression of the channel to the brain, suggests the application of this protein for diagnostic or therapeutic purposes.
| S-EPMC1618397 | biostudies-literature
Project description:Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates cardiac action potential repolarization. Drugs that activate hERG1 channels represent a mechanism-based approach for the treatment of long QT syndrome, a disorder of cardiac repolarization associated with ventricular arrhythmia and sudden death. Here, we characterize the mechanisms of action and the molecular determinants for binding of RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR), a recently discovered hERG1 channel activator. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured by using the two-microelectrode voltage-clamp technique. RPR induced a concentration-dependent slowing in the rate of channel deactivation and enhanced current magnitude by shifting the voltage dependence of inactivation to more positive potentials. This mechanism was confirmed by demonstrating that RPR slowed the rate of deactivation, but did not increase current magnitude of inactivation-deficient mutant channels. The effects of RPR on hERG1 kinetics and magnitude could be simulated by reducing three rate constants in a Markov model of channel gating. Point mutations of specific residues located in the S4-S5 linker or cytoplasmic ends of the S5 and S6 domains greatly attenuated or ablated the effects of 3 microM RPR on deactivation (five residues), inactivation (one residue), or both gating mechanisms (four residues). These findings define a putative binding site for RPR and confirm the importance of an interaction between the S4-S5 linker and the S6 domain in electromechanical coupling of voltage-gated K(+) channels.
| S-EPMC1959467 | biostudies-literature
Project description:Drug induced long QT syndrome is a high risk event in clinic, which mainly results from their high affinity to the Human Ether-a-go-go-Related Gene (hERG) potassium channel. Therefore, evaluation of the drug's inhibitory activity against the hERG potassium channel is a required step in drug discovery and development. In this study, we developed a series of novel conformation-mediated intramolecular photoinduced electron transfer fluorogenic probes for the hERG potassium channel. After careful evaluation, probes N4 and N6 showed good activity and may have a promising application in the cell-based hERG potassium channel inhibitory activity assay, as well as potential hERG-associated cardiotoxicity evaluation. Compared with other assay methods, such as patch clamp assay, radio-ligand competitive binding assay, fluorescence polarization and potential-sensitive fluorescent probes, this method is convenient and can also selectively measure the inhibitory activity in the native state of the hERG potassium channel. Meanwhile, these probes can also be used for hERG potassium channel imaging without complex washing steps.
| S-EPMC4658274 | biostudies-literature
Project description:The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.
| S-EPMC5034037 | biostudies-literature
Project description:Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers.
| S-EPMC4127929 | biostudies-literature
Project description:The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism ∼11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac electrophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (I Kr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of I Kr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.
| S-EPMC6322893 | biostudies-literature
Project description:AIM:To study the expression of ether à go-go (Eag1) potassium channel in colorectal cancer and the relation-ship between their expression and clinico-pathological features. METHODS:The expression levels of Eag1 protein were determined in 76 cancer tissues with paired non-cancerous matched tissues as well as 9 colorectal adenoma tissues by immunohistochemistry. Eag1 mRNA expression was detected in 13 colorectal cancer tissues with paired non-cancerous matched tissues and 4 colorectal adenoma tissues as well as two colorectal cancer cell lines (LoVo and HT-29) by reverse transcription PCR. RESULTS:The frequency of positive expression of Eag1 protein was 76.3% (58/76) and Eag1 mRNA was 76.9% (10/13) in colorectal cancer tissue. Expression level of Eag1 protein was dependent on the tumor size, lymphatic node metastasis, other organ metastases and Dukes' stage (P < 0.05), while not dependent on age, sex, site and degree of differentiation. Eag1 protein and mRNA were negative in normal colorectal tissue, and absolutely negative in colorectal adenomas except that one case was positively stained for Eag1 protein. CONCLUSION:Eag1 protein and mRNA are aberrantly expressed in colorectal cancer and occasionally expressed in colorectal adenoma. The high frequency of expression of Eag1 in tumors and the restriction of normal expression to the brain suggest the potential of this protein for diagnostic, prognostic and therapeutic purposes.
| S-EPMC4147004 | biostudies-literature
Project description:The detection and imaging of hERG potassium channels in living cells can provide useful information for hERG-correlation studies. Herein, three small-molecule fluorescent probes, based on the potent hERG channel inhibitor astemizole, for the imaging of hERG channels in hERG-transfected HEK293 cells (hERG-HEK293) and human colorectal cancer cells (HT-29), are described. These probes are expected to be applied in the physiological and pathological studies of hERG channels.
| S-EPMC4789673 | biostudies-literature