Molecular determinants of pentamidine-induced hERG trafficking inhibition.
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ABSTRACT:
SUBMITTER: Dennis AT
PROVIDER: S-EPMC3263949 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 ?M R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine's relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.
| S-EPMC6719874 | biostudies-literature
Project description:The alpha subunit of the voltage gated human ether-a-go-go-related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. HERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Hypoxia, a common feature of tumors, alters gating properties of hERG currents in SH-SY5Y neuroblastoma cells. In the present study, we examined the molecular mechanisms and physiological significance underlying hypoxia-altered hERG currents in SH-SY5Y neuroblastoma cells. Hypoxia reduced the surface expression of 150kDa form and increased 125kDa form of hERG protein expression in the endoplasmic reticulum (ER). The changes in protein expression were associated with ~50% decrease in hERG potassium conductance. ER retention of hERG 125kDa form by CH was due to defective trafficking and was rescued by exposing cells to hypoxia at low temperatures or treatment with E-4031, a hERG channel blocker. Prolonged association of hERG with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contributed to ER accumulation and trafficking defect. Hypoxia increased reactive oxygen species (ROS) levels and manganese (111) tetrakis (1methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant prevented hypoxia-induced degradation of 150kDa and accumulation of 125kDa forms. Impaired trafficking of hERG by hypoxia was associated with reduced cell proliferation and this effect was prevented by antioxidant treatment. These results demonstrate that hypoxia through increased oxidative stress impairs hERG trafficking, leading to decreased K+ currents resulting in cell cycle arrest in SH-SY5Y cells.
| S-EPMC6481834 | biostudies-literature
Project description:Clinically, drug-induced torsades de pointes (TdP) are rare events, whereas the reduction of the human ether-à-go-go-related gene (hERG) current is common. In this study, we aimed to explore the specific factors that contribute to the deterioration of hERG inhibition into malignant ventricular arrhythmias. Cisapride, a drug removed from the market because it caused long QT (LQT) syndrome and torsade de pointes (TdP), was used to induce hERG inhibition. The effects of cisapride on the hERG current were evaluated using a whole-cell patch clamp. Based on the dose-response curve of cisapride, models of its effects at different doses (10, 100, and 1,000 nM) on guinea pig heart in vitro were established. The effects of cisapride on electrocardiogram (ECG) signals and QT interval changes in the guinea pigs were then comprehensively evaluated by multi-channel electrical mapping and high-resolution fluorescence mapping, and changes in the action potential were simultaneously detected. Cisapride dose-dependently inhibited the hERG current with a half inhibitory concentration (IC50) of 32.63 ± 3.71 nM. The complete hERG suppression by a high dose of cisapride (1,000 nM) prolonged the action potential duration (APD), but not early after depolarizations (EADs) and TdP occurred. With 1 μM cisapride and lower Mg2+/K+, the APD exhibited triangulation, dispersion, and instability. VT was induced in two of 12 guinea pig hearts. Furthermore, the combined administration of isoproterenol was not therapeutic and increased susceptibility to ventricular fibrillation (VF) development. hERG inhibition alone led to QT and ERP prolongation and exerted an anti-arrhythmic effect. However, after the combination with low concentrations of magnesium and potassium, the prolonged action potential became unstable, triangular, and dispersed, and VT was easy to induce. The combination of catecholamines shortened the APD, but triangulation and dispersion still existed. At this time, VF was easily induced and sustained.
| S-EPMC9354841 | biostudies-literature
Project description:In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.
| S-EPMC5393917 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. EXPERIMENTAL APPROACH: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. KEY RESULTS: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.
| S-EPMC2241789 | biostudies-other
Project description:The primate-specific brain voltage-gated potassium channel isoform Kv11.1-3.1 has been identified as a novel therapeutic target for the treatment of schizophrenia. While this ether-a-go-go related K(+)channel has shown clinical relevance, drug discovery efforts have been hampered due to low and inconsistent activity in cell-based assays. This poor activity is hypothesized to result from poor trafficking via the lack of an intact channel-stabilizing Per-Ant-Sim (PAS) domain. Here we characterize Kv11.1-3.1 cellular localization and show decreased channel expression and cell surface trafficking relative to the PAS-domain containing major isoform, Kv11.1-1A. Using small molecule inhibition of proteasome degradation, cellular expression and plasma membrane trafficking are rescued. These findings implicate the importance of the unfolded-protein response and endoplasmic reticulum associated degradation pathways in the expression and regulation of this schizophrenia risk factor. Utilizing this identified phenomenon, an electrophysiological and high throughput in-vitro fluorescent assay platform has been developed for drug discovery in order to explore a potentially new class of cognitive therapeutics.
| S-EPMC4754628 | biostudies-literature
Project description:ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.
| S-EPMC1170667 | biostudies-other
Project description:We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 microM. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 microM) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10- to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that intron-containing strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.
| S-EPMC89798 | biostudies-literature
Project description:BackgroundPilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr ) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system.MethodsHEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments.ResultsAcute exposure to pilsicainide at 0.03-10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03-10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W.ConclusionsPilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.
| S-EPMC5459418 | biostudies-literature
Project description:A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.
| S-EPMC3174182 | biostudies-literature