Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.
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ABSTRACT:
SUBMITTER: Wang C
PROVIDER: S-EPMC4816235 | biostudies-other | 2015 Dec
REPOSITORIES: biostudies-other
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Project description:The human ether-a-go-go-related gene (hERG) potassium channel conducts rapid delayed rectifier potassium currents (IKr) and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR) has multiple actions, and its hydrogenated derivative dihydroberberine (DHB) is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD). Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652) and phenylalanine (Phe656), and decreased mature (155-kDa) and simultaneously increased immature (135-kDa) hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90) expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6), a biomarker of unfolded protein response (UPR). Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was disrupted by impaired channel folding associated with altered interactions between hERG proteins and chaperones. Finally, trafficking inhibition activated UPR, and mature hERG channel degradation was increased by DHB.
| S-EPMC5538702 | biostudies-literature
Project description:Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG) channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells), and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC50 of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV). Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea) or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively). Green tea Lung Ching and red wine inhibited hERG currents, with IC50 of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities.
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Project description:The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.
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Project description:L-type calcium currents (I(Ca)) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I(Ca) and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca(2+) channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from approximately 75%-80% to approximately 50% by omitting beta subunits but unaffected by omitting alpha(2)delta subunits. Similarly, gluconate inhibition was reduced to approximately 50% by deleting an alpha1 subunit N-terminal region of 15 residues critical for beta subunit interactions regulating open probability. Omitting beta subunits with this mutant alpha1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different beta subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from approximately 75%-80% to approximately 50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to approximately 60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to approximately 25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving beta subunit interactions with the N terminus and a short C terminal region.
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Project description:Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels.Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2?36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software.The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 ?mol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 ?mol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2?36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered.Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits.
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Project description:(1) We have determined the molecular basis of nicardipine-induced block of cardiac transient outward K(+) currents (I(to)). Inhibition of I(to) was studied using cloned voltage-dependent K(+) channels (Kv) channels, rat Kv4.3L, Kv4.2, and Kv1.4, expressed in human embryonic kidney cell line 293 (HEK293) cells. (2) Application of the dihydropyridine Ca(2+) channel antagonist, nicardipine, accelerated the inactivation rate and reduced the peak amplitude of Kv4.3L currents in a concentration-dependent manner (IC(50): 0.42 micro M). The dihydropyridine (DHP) Ca(2+) channel agonist, Bay K 8644, also blocked this K(+) current (IC(50): 1.74 micro M). (3) Nicardipine (1 micro M) slightly, but significantly, shifted the voltage dependence of activation and steady-state inactivation to more negative potentials, and also slowed markedly the recovery from inactivation of Kv4.3L currents. (4) Coexpression of K(+) channel-interacting protein 2 (KChIP2) significantly slowed the inactivation of Kv4.3L currents as expected. However, the features of DHP-induced block of K(+) current were not substantially altered. (5) Nicardipine exhibited similar block of Kv1.4 and Kv4.2 channels stably expressed in HEK293 cells; IC(50)'s were 0.80 and 0.62 micro M, respectively. (6) Thus, at submicromolar concentrations, DHP Ca(2+) antagonist and agonist inhibit Kv4.3L and have similar inhibiting effects on other components of cardiac I(to), Kv4.2 and Kv1.4.
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Project description:PurposeTo quantify the affinity of a cationic computed tomography (CT) contrast agent (CA(4+)) and that of an anionic contrast agent (ioxaglate) to glycosaminoglycans (GAGs) in ex vivo cartilage tissue explants and to characterize the in vivo diffusion kinetics of CA(4+) and ioxaglate in a rabbit model.Materials and methodsAll in vivo procedures were approved by the institutional animal care and use committee. The affinities of ioxaglate and CA(4+) to GAGs in cartilage (six bovine osteochondral plugs) were quantified by means of a modified binding assay using micro-CT after plug equilibration in serial dilutions of each agent. The contrast agents were administered intraarticularly to the knee joints of five New Zealand white rabbits to determine the in vivo diffusion kinetics and cartilage tissue imaging capabilities. Kinetics of diffusion into the femoral groove cartilage and relative contrast agent uptake into bovine plugs were characterized by means of nonlinear mixed-effects models. Diffusion time constants (τ) were compared by using a Student t test.ResultsThe uptake of CA(4+) in cartilage was consistently over 100% of the reservoir concentration, whereas it was only 59% for ioxaglate. In vivo, the contrast material-enhanced cartilage reached a steady CT attenuation for both CA(4+) and ioxaglate, with τ values of 13.8 and 6.5 minutes, respectively (P = .04). The cartilage was easily distinguishable from the surrounding tissues for CA(4+) (12 mg of iodine per milliliter); comparatively, the anionic contrast agent provided less favorable imaging results, even when a higher concentration was used (80 mg of iodine per milliliter).ConclusionThe affinity of the cationic contrast agent CA(4+) to GAGs enables high-quality imaging and segmentation of ex vivo bovine and rabbit cartilage, as well as in vivo rabbit cartilage.Supplemental materialhttp://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112246/-/DC1.
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Project description:Transcatheter therapies are emerging for functional mitral regurgitation (FMR) treatment, however there is lack of pathological models for their preclinical assessment. We investigated the applicability of deer hearts for this purpose.8 whole deer hearts were housed in a pulsatile flow bench. At baseline, all mitral valves featured normal coaptation. The pathological state was induced by 60-minutes intraventricular constant pressurization. It caused mitral annulus dilation (antero-posterior diameter increase from 31.8 ± 5.6 mm to 39.5 ± 4.9 mm, p = 0.001), leaflets tethering (maximal tenting height increase from 7.3 ± 2.5 mm to 12.7 ± 3.4 mm, p < 0.001) and left ventricular diameter increase (from 67.8 ± 7.5 mm to 79.4 ± 6.5 mm, p = 0.004). These geometrical reconfigurations led to restricted mitral valve leaflets motion and leaflet coaptation loss. Preliminary feasibility assessment of two FMR treatments was performed in the developed model.Deer hearts showed ability to dilate under constant pressurization and have potential to be used for realistic preclinical research of novel FMR therapies. Graphical abstract figure legend: Deer heart mitral valve fiberscopic and echocardiographic images in peak systole at baseline and after inducing the pathological conditions representing functional mitral regurgitation. In the pathological conditions lack of coaptation between the leaflets, enlargement of the antero-posterior distance (red dashed line) and the left ventricular diameter (orange dashed line) were observed.
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Project description:BACKGROUND AND PURPOSE: Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions, the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. Here we report that this compound exerts non-specific inhibitory actions on the TRPC6 channel, a transient receptor potential (TRP) protein. EXPERIMENTAL APPROACH: Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques. KEY RESULTS: Cationic currents elicited by carbachol (CCh; 100 microM) in HEK293 cells overexpressing murine TRPC6 (I(TRPC6)) were dose-dependently inhibited by externally applied ML-9 (IC(50)=7.8 microM). This inhibition was voltage-dependent and occurred as fast as external Na(+) removal. Another MLCK inhibitor, wortmannin (3 microM), and MLCK inhibitory peptides MLCK-IP(11-19) (10 microM) and -IP(480-501) (1 microM) showed little effects on I(TRPC6) density and the inhibitory efficacy of ML-9. The extent of the inhibition also unchanged with co-expression of wild-type or a dominant negative mutant of MLCK. Inhibitory effects of ML-9 on I(TRPC6) remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 microM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed I(TRPC6). In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished approximately 35pS single TRPC6 channel activities. Contrarily, currents due to TRPC7 expression were rapidly enhanced by externally applied ML-9 and ML-7, which was not prevented by MLCK inhibitory peptides. CONCLUSION AND IMPLICATIONS: These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity.
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Project description:Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from ?-sarcoglycan-null and laminin-?(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.
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