Inhibition Of Channel Currents Research Articles

Acacetin is a Promising Drug Candidate for Cardiovascular Diseases.

Phytochemical flavonoids have been proven to be effective in treating various disorders, including cardiovascular diseases. Acacetin is a natural flavone with diverse pharmacological effects, uniquely including atrial-selective anti-atrial fibrillation (AF) via the inhibition of the atrial specific potassium channel currents [Formula: see text] (ultra-rapidly delayed rectifier potassium current), [Formula: see text] (acetylcholine-activated potassium current), [Formula: see text] (calcium-activated small conductance potassium current), and [Formula: see text] (transient outward potassium current). [Formula: see text] inhibition by acacetin, notably, suppresses experimental J-wave syndromes. In addition, acacetin provides extensive cardiovascular protection against ischemia/reperfusion injury, cardiomyopathies/heart failure, autoimmune myocarditis, pulmonary artery hypertension, vascular remodeling, and atherosclerosis by restoring the downregulated intracellular signaling pathway of Sirt1/AMPK/PGC-1[Formula: see text] followed by increasing Nrf2/HO-1/SOD thereby inhibiting oxidation, inflammation, and apoptosis. This review provides an integrated insight into the capabilities of acacetin as a drug candidate for treating cardiovascular diseases, especially atrial fibrillation and cardiomyopathies/heart failure.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Modulation of Kv7 Channel Currents by Echinocystic Acid.

Modulation of KCNQ-encoded voltage-gated potassium Kv7/M channel function represents an attractive strategy to treat neuronal excitability disorders such as epilepsy, pain, and depression. The Kv7 channel group includes five subfamily members (Kv7.1-7.5). Pentacyclic triterpenes display extensive pharmacological activities including anti-tumor, anti-inflammatory, and anti-depression effects. In this study, we investigated the effects of pentacyclic triterpenes on Kv7 channels. Our results show that echinocystic acid, ursonic acid, oleanonic acid, demethylzeylasteral, corosolic acid, betulinaldehyde, acetylursolic acid, and ⍺-boswellic acid gradually exert decreasing degrees of Kv7.2/Kv7.3 channel current inhibition. Echinocystic acid was the most potent inhibitor, with a half-maximal inhibitory concentration (IC50) of 2.5 µM. It significantly shifted the voltage-dependent activation curve in a positive direction and slowed the time constant of activation for Kv7.2/Kv7.3 channel currents. Furthermore, echinocystic acid non-selectively inhibited Kv7.1-Kv7.5 channels. Taken together, our findings indicate that echinocystic acid is a novel and potent inhibitor that could be used as a tool to further understand the pharmacological functions of neuronal Kv7 channels. Significance Statement Pentacyclic triterpenes reportedly have multiple potential therapeutic uses such as anticancer, anti-inflammatory, antioxidant, and anti-depression effects. In the present study, we show that echinocystic acid, ursonic acid, oleanonic acid, and demethylzeylasteral inhibit Kv7.2/Kv7.3 channels to varying degrees. Of these, echinocystic acid was the most potent Kv7.2/Kv7.3 current inhibitor and inhibited Kv7.1-Kv7.5 currents in a non-selective manner.

Tmc regulates membrane viscosity in mammalian cochlear hair cells.

The hair bundle, composed of rows of actin-filled stereocilia, is the sensory organelle of inner ear hair cells. There is a limited but growing body of data suggesting that stereocilia membrane properties modulate mechanically-gated (MET) ion channels located on the stereocilia. Transmembrane Channel Like proteins (TMCs) are considered part of the MET channel machinery, but also have membrane scramblase activity. To further investigate the role of membrane in modulating MET channel and test whether the presence of TMCs might account for the previously reported differences in stereocilia and soma membrane properties, we used a novel viscosity sensor BODIPY1c. The lifetime changes of BODIPY1c based on viscosity allowed precise spatial and dynamic monitoring of membrane properties within live hair cells. BODIPY1c can also enter hair cells through MET channels and fluorescently label the cytoplasmic membranes, thus allowing to identify hair cells with functional MET channels. Using synthesized liposomes of sizes ranging from 90-2000 nm with lipid composition matching that of stereocilia, we show that membrane curvature established with stereocilia morphology will not account for the observed difference between stereocilia and soma. We also show that the membrane viscosity of stereocilia and soma of mammalian cochlear hair cells vary during development. Membrane viscosity decreases and strongly correlates with the onset of MET. TMIE and TMC1 mutant mice, both of which lack TMC1 in stereocilia, have significantly higher membrane viscosity compared to litter mate controls at P10. In TMC1 mutants, the stereocilia membrane viscosity strongly correlates to the level at which the bundles are transducing. Inhibition of MET channel current in P10 rats for 30 mins with 1 mM curare, however, did not alter the membrane viscosity suggesting that the MET current is not directly driving the decrease in membrane viscosity.

Intracellular uptake of agents that block the hERG channel can confound the assessment of QT interval prolongation and arrhythmic risk.

Oliceridine is a biased ligand at the μ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 μM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.

Diabetes and SGLT2-iss inhibitors in patients with heart failure with preserved or mid-range left ventricular ejection fractions.

Diabetic patients frequently develop heart failure with preserved (HFpEF) or mid-range (HFmEF) cardiac ejection fractions. This condition may be secondary to diabetic cardiomyopathy or one of several relevant comorbidities, mainly hypertension. Several mechanisms link diabetes to HFpEF or HFmEF. Among these, non-enzymatic glycation of interstitial proteins, lipotoxicity, and endothelial dysfunction may promote structural damage and ultimate lead to heart failure. Findings from several large-scale trials indicated that treatment with sodium/glucose cotransporter 2 inhibitors (SGLT2-iss) resulted in significant improvements in cardiovascular outcomes in diabetic patients with high cardiovascular risk. However, there is currently some evidence that suggests a clinical advantage of using SGLT2-iss specifically in cases of HFpEF or HFmEF. Preclinical and clinical studies revealed that SGLT2-iss treatment results in a reduction in left ventricular mass and improved diastolic function. While some of the beneficial effects of SGLT2-iss have already been characterized (e.g., increased natriuresis and diuresis as well as reduced blood pressure, plasma volume, and arterial stiffness, and nephron-protective activities), there is increasing evidence suggesting that SGLT2-iss may have direct actions on the heart. These findings include SGLT2-iss-mediated reductions in the expression of hypertrophic foetal genes and diastolic myofilaments stiffness, increases in global phosphorylation of myofilament regulatory proteins (in HFpEF), inhibition of cardiac late sodium channel current and Na+/H+ exchanger activity, metabolic shifts, and effects on calcium cycling. Preliminary data from previously published studies suggest that SGLT2-iss could be useful for the treatment of HFpEF and HFmEF. Several large ongoing trials, including DELIVER AND EMPEROR -preserved have been designed to evalute the efficacy of SGLT2-iss in improving clinical outcomes in patients diagnosed with HFpEF. The goal of this manuscript is to review the use of SGLT2-iss inhibitors for HFpEF or HFmEF associated with diabetes.

Safety pharmacology of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle platform.

The present safety pharmacology core battery studies (neurobehavior, respiratory, cardiovascular system, and human ether a-go-go (hERG) channel current) investigated the potential harmful effects of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG). The SAMiRNA-AREG was administered by single intravenous injection at up to 300 mg/kg and 100 mg/kg in mice and monkeys, respectively. The hERG assay was performed in Chinese hamster ovary (CHO) cells at SAMiRNA-AREG concentrations of up to 200 μg/mL. In the evaluation on neurobehavior, a transient decrease in body temperature was found at 0.5 h (30 min) post-dose at both sexes in mice, with a single 300 mg/kg dose of SAMiRNA-AREG. However, these effects had returned to normal at 1 h post-dose. In the evaluation on hERG channel current, there were statistically significant differences in the inhibition of peak hERG potassium channel current between the 20, 100, and 200 μg/mL SAMiRNA-AREG treatment groups and the vehicle control group. However, these effects were less potent than that of E-4031, a positive control article. For the respiratory and cardiovascular systems, no treatment-related changes were observed in mice or monkeys. Thus, under these experimental conditions, these studies suggest that SAMiRNA-AREG showed no adverse effects on the neurobehavior, respiratory, and cardiovascular function.

Inhibition of CRAC channel current by TRPV1 channel agonist and antagonist in RBL cells

Inhibition of CRAC channel current by TRPV1 channel agonist and antagonist in RBL cells

Inhibition of warm temperature‐sensitive and Ca2+‐permeable TRPV3 channel by a novel compound QDULM‐12 for anti‐pruritus and dermatitis

Transient receptor potential vanilloid 3 (TRPV3) channel as a member of thermoTRPV subfamily is primarily expressed in the keratinocytes of the skin and sensory neurons. Genetic gain‐of‐function mutations of TRPV3 gene cause skin‐related diseases such as pruritus and dermatitis, indicating that pharmacological inhibition of overactive TRPV3 may be beneficial for skin health and diseases. However, there is a significant lack of specific tools that can be used to validate TRPV3 channel as therapeutic target for skin diseases or indications yet to be defined. Recently, we made a serial modifications on natural bioactive compound and evaluated their inhibitory activities on human TRPV3 channels expressed in HEK293 cells using whole‐cell patch clamp recordings. We identified a hit compound QDULM‐12 that exhibited a dose‐dependent inhibition of hTRPV3 channel current activated by agonist 2‐APB (50 μM) with an IC50 value in a range of 0.1 μM. The selectivity of QDULM‐12 compound over other thermoTRP channels and its biological effects on in vitro keratinocytes and in vivo pruritus and dermatitis are currently underway.Support or Funding InformationThis work was supported by research grants awarded to XYS from the National Natural Sciences Foundation of China (81903734), the Natural Science Foundation of Shandong Province (ZR2017BH020), and to WKW from the National Natural Sciences Foundation of China (81573410), the Ministry of Science and Technology of China (2018ZX09711001‐004‐006).

EAG Channel PAS Domain Binder Inhibits Currents from EAG Channels and Decreases Tumor Growth in Zebrafish Xenograft Model

Ether-a-go-go (EAG) potassium selective channels regulate neuronal excitability and tumorigenesis. Inhibition of EAG channel currents has been shown to decrease cancer progression. EAG channels contain a Per-Arn-Sim (PAS) domain in their N-terminal region. The PAS domain is structurally similar to the PAS domains in other non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, small molecule binders of the EAG PAS domain have not been identified. Using surface-plasmon resonance (SPR) based high-throughput screen we identified an EAG PAS domain small molecule binder which binds to the PAS domain in a concentration dependent manner. Application of the identified small molecule binder to the excised inside-out membrane patches expressing EAG channels inhibited currents recorded from EAG channels with an IC50 of 4.09 + 0.61 μM. Deletion of the PAS domain dramatically decreased the inhibition of EAG channel currents by the identified small molecule ligand, indicating that the effect on EAG currents is directly caused by binding of the ligand to the PAS domain. To investigate the therapeutic potential of the identified ligand we used zebrafish xenograft model. Treatment with the identified ligand decreased tumor growth in zebrafish implanted with cell lines known to overexpress EAG channels, while had statistically insignificant effect on tumor growth in zebrafish implanted with a cell line that does not overexpress EAG channels. Taken together, our study indicates that the PAS domain can function as a ligand-binding domain in EAG channels. Importantly, the identified ligand is an FDA approved drug. Thus, our study presents a case for repurposing the identified ligand for cancer treatment as an EAG channel inhibitor.

Anti-Nociceptive and Anti-Inflammation Effect Mechanisms of Mutants of Syb-prII, a Recombinant Neurotoxic Polypeptide.

Syb-prII, a recombinant neurotoxic polypeptide, has analgesic effects with medicinal value. Previous experiments indicated that Syb-prII displayed strong analgesic activities. Therefore, a series of in vivo and vitro experiments were designed to investigate the analgesic and anti-inflammatory properties and possible mechanisms of Syb-prII. The results showed that administered Syb-prII-1 and Syb-prII-2 (0.5, 1, 2.0 mg/kg, i.v.) to mice significantly reduced the time of licking, biting, or flicking of paws in two phases in formalin-induced inflammatory nociception. Syb-prII-1 inhibited xylene-induced auricular swelling in a dose-dependent manner. The inhibitory effect of 2.0 mg/kg Syb-prII-1 on the ear swelling model was comparable to that of 200 mg/kg aspirin. In addition, the ELISA and Western blot analysis suggested that Syb-prII-1 and Syb-prII-2 may exert an analgesic effect by inhibiting the expression of Nav1.8 and the phosphorylation of ERK, JNK, and P38. Syb-prII-1 markedly suppressed the expression of IL-1β, IL-6, and TNF-α of mice in formalin-induced inflammatory nociception. We used the patch-clamp technique and investigated the effect of Syb-prII-1 on TTX-resistant sodium channel currents in acutely isolated rat DRG neurons. The results showed that Syb-prII-1 can significantly down regulate TTX-resistant sodium channel currents. In conclusion, Syb-prII mutants may alleviate inflammatory pain by significantly inhibiting the expression of Nav1.8, mediated by the phosphorylation of MAPKs and significant inhibition of TTX-resistant sodium channel currents.

Identification of a single aspartate residue critical for both fast and slow calcium-dependent inactivation of the human TRPML1 channel

Transient receptor potential mucolipin subfamily 1 (TRPML1) is a nonselective cation channel mainly located in late endosomes and lysosomes. Mutations of the gene encoding human TRPML1 can cause severe lysosomal diseases. The activity of TRPML1 is regulated by both Ca2+ and H+, which are important for its critical physiological functions in membrane trafficking, exocytosis, autophagy, and intracellular signal transduction. However, the molecular mechanism of its dual regulation by Ca2+ and H+ remains elusive. Here, using a mutant screening method in combination with a whole-cell patch clamp technique, we identified a key TRPML1 residue, Asp-472, responsible for both fast calcium-dependent inactivation (FCDI) and slow calcium-dependent inactivation (SCDI) as well as H+ regulation. We also found that, in acidic pH, H+ can significantly delay FCDI and abolish SCDI and thereby presumably facilitate the ion conductance of the human TRPML1 channel. In summary, we have identified a key residue critical for Ca2+-induced inhibition of TRPML1 channel currents and uncovered pH-dependent regulation of this channel, providing vital information regarding the detailed mechanism of action of human TRPML1.

Glyco-metabolic Effects of Ranolazine: A truly Multifaceted Drug?

Ranolazine (R) is a non-haemodynamic anti-anginal agent used as adjunctive therapy in patients with chronic coronary heart disease (CHD) whose symptoms are unadequately controlled by conventional treatment. R decreases calcium overload in the ischemic myocytes through the inhibition of late sodium channel current in the myocardium; induces myocardial relaxation and improves myocardial blood flow. In addition to its anti-angina effect, other possible clinical applications of R have also been explored, including treatment of atrial arrhythmias, chronic heart failure and diabetes. With regard to diabetes R has been shown to significantly improve glycemic control in diabetic patients with CHD in post-hoc analysis of large-scale clinical trials. Moreover reduction of glycated hemoglobin and fasting serum glucose levels have been also observed prospectively in small clinical trials conducted on diabetic subjects without CHD by using R alone or in combination with other oral glucose-lowering drugs. Lastly R improved insulin resistance in non-diabetic overweight/obese patients with CHD. Hypothetical mechanisms of this metabolic action are: inhibition of secretion of glucagon from pancreatic islets, preservation of beta cells and increase of insulin delivery to tissues. In light of these observations R appear to be a potential multifaceted drug that offers the opportunity to treat glyco-metabolic disorders alone or in association to CHD. However, prospective studies are too small to be conclusive and new trials are needed to clarify which is the exact role of R in the treatment of glyco-metabolic disorders.

Real Patient and its Virtual Twin: Application of Quantitative Systems Toxicology Modelling in the Cardiac Safety Assessment of Citalopram

A quantitative systems toxicology (QST) model for citalopram was established to simulate, in silico, a ‘virtual twin’ of a real patient to predict the occurrence of cardiotoxic events previously reported in patients under various clinical conditions. The QST model considers the effects of citalopram and its most notable electrophysiologically active primary (desmethylcitalopram) and secondary (didesmethylcitalopram) metabolites, on cardiac electrophysiology. The in vitro cardiac ion channel current inhibition data was coupled with the biophysically detailed model of human cardiac electrophysiology to investigate the impact of (i) the inhibition of multiple ion currents (IKr, IKs, ICaL); (ii) the inclusion of metabolites in the QST model; and (iii) unbound or total plasma as the operating drug concentration, in predicting clinically observed QT prolongation. The inclusion of multiple ion channel current inhibition and metabolites in the simulation with unbound plasma citalopram concentration provided the lowest prediction error. The predictive performance of the model was verified with three additional therapeutic and supra-therapeutic drug exposure clinical cases. The results indicate that considering only the hERG ion channel inhibition of only the parent drug is potentially misleading, and the inclusion of active metabolite data and the influence of other ion channel currents should be considered to improve the prediction of potential cardiac toxicity. Mechanistic modelling can help bridge the gaps existing in the quantitative translation from preclinical cardiac safety assessment to clinical toxicology. Moreover, this study shows that the QST models, in combination with appropriate drug and systems parameters, can pave the way towards personalised safety assessment.

Acute and chronic effects of taurine magnesium coordination compound on cardiac sodium channel Nav1.5.

It has been previously demonstrated that taurine magnesium coordination compound (TMCC) produces antiarrhythmic effects invivo. The present study investigated the acute and chronic effect of TMCC on sodium channels in HEK cells stably expressing human cardiac Nav1.5 sodium channels. The current amplitude, activation and inactivation kinetics, recovery time from inactivation, and use‑dependent block of sodium channels were analyzed using the whole‑cell patch clamp technique. Western blotting was used to analyze Nav1.5 expression following chronic TMCC treatment. In HEK cells expressing Nav1.5 channels, TMCC acutely inhibited Na+ currents in a dose‑dependent manner. In addition, acute application of TMCC shifted the activation and inactivation curves, and prolonged the recovery time from inactivation, but did not exhibit a use‑dependent block of Nav1.5. By contrast, chronic TMCC treatment only produced a use‑dependent block of Nav1.5 and downregulated Nav1.5 expression. The results of the present study suggested that TMCC may produce antiarrhythmic actions via acute inhibition of sodium channel currents and chronic downregulation of Nav1.5 expression.

Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors

κ-Hexatoxins (κ-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic κ-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BKCa or KCa1.1) channels, but not their mammalian orthologs. Despite this potent and selective action on insect KCa1.1 channels, we found that the classical KCa1.1 blockers paxilline, charybdotoxin and iberiotoxin, which all block insect KCa1.1 channels, are not lethal in crickets. We therefore used whole-cell patch-clamp analysis of cockroach dorsal unpaired median (DUM) neurons to study the effects of κ-HXTX-Hv1c on sodium-activated (KNa), delayed-rectifier (KDR) and ‘A-type’ transient (KA) K+ channels. 1 μM κ-HXTX-Hv1c failed to significantly inhibit cockroach KNa and KDR channels, but did cause a 30 ± 7% saturating inhibition of KA channel currents, possibly via a Kv4 (Shal-like) action. However, this modest action at such a high concentration of κ-HXTX-Hv1c would indicate a different lethal target. Accordingly, we assessed the actions of κ-HXTX-Hv1c on neurotransmitter-gated ion channels in cockroach DUM neurons. We found that κ-HXTX-Hv1c failed to produce any major effects on GABAA or glutamate-Cl receptors but dramatically slowed nicotine-evoked ACh receptor (nAChR) current decay and reversed nAChR desensitization. These actions occurred without any alterations to nAChR current amplitude or the nicotine concentration-response curve, and are consistent with a positive allosteric modulation of nAChRs. κ-HXTX-Hv1c therefore represents the first venom peptide that selectively modulates insect nAChRs with a mode of action similar to the excitotoxic insecticide spinosyn A.This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’

Abstract P273: Increased Inhibition of Potassium Channel Currents by Angiotensin II in Sympathetic Neurons may Contribute to a Sustained Blood Pressure Elevation in (mRen2)27 Rats.

It is well established that the increased sympathetic tone may contribute to initiation and progression of various forms of hypertension. Several lines of evidence suggest a link between the renin-angiotensin system and sympathetic nerve activity in hypertension, and the previous studies in animal models demonstrated increased sympathetic output in the presence of Angiotensin II (AngII). To elucidate potential underlying molecular mechanisms of such phenomenon, we compared the effect of AngII on the whole-cell potassium channel currents in superior cervical ganglia (SCG) neurons isolated from hypertensive (mRen2)27 rats with overexpression of renin gene, and control Sprague Dawley® (SD) rats. In both groups, the whole-cell potassium channel currents were identified as rapidly-activating, 4-Aminopyridine-sensitive transient A-type currents, as well as slowly-activating tetraethylammonium-sensitive delayed rectifier currents. When the cell membrane was depolarized to -40, -30 and -20 mV from a holding potential of -80 mV, AngII (100 nM) profoundly inhibited A-type current, but the magnitude of such inhibition was not significantly different between neurons isolated from (mRen2)27 (38.1±6.2%, 47.8±5.7% and 52.1±5.7%; n=11) and SD rats (37.2±4.6%, 44.±4.5% and 46.1±4.8%; n = 13). Delayed rectifier potassium channel currents were isolated by holding cells at -40 mV, which resulted in complete elimination of the transient A-type current. In contrast to transient current, inhibition of the delayed rectifier current by AngII in the range of membrane potentials between +20 and +80 mV was significantly greater (p<0.05) in neurons obtained from (mRen2)27 rats (11.0±3.2% to 25.0±2.9%, n=12) when compared to SD rats (4.7±1.5% to 16.3±2.7%, n = 12). In both groups, inhibition of both channel types was completely abolished by 10 uM Losartan, indicating involvement of AT1 receptors. Our results suggest that in (mRen2)27 hypertensive rats, the increased inhibitory effect of AngII on delayed rectifier potassium channel currents could possibly lead to lowering spike threshold, which, in turn, could elevate sympathetic outflow and lead to sustained blood pressure elevation.

Identification of the first in silico-designed TREK1 antagonists that block channel currents dose dependently.

TREK1 (Twik-RElated Potassium (K+ ) channel 1), although a well-characterized target for several neuropsychiatric disorders, underwent very few explorations for prototypic inhibitors. This study aimed to find diverse chemotypes by an in silico means. Homology-built TREK1 on docking with high-affinity quaternary ammonium compounds (QAs) corroborated the previous findings by recreating the binding mode with proximally positioned key residues: Thr157, Thr266, Ile182, Leu189, and Leu304. Physical interactions between TREK1 and known antagonists were modeled to compensate the lack of ligand-bound protein crystal structures. A common feature hypothesis (Hypo1) was deduced from the chemical features of six active compounds. Validated Hypo1 and the most potent compound in the data set were employed as pharmacophore- and similarity-based virtual screening queries, respectively. Thirty-three hit compounds were tested for their ability to block TREK1 currents in HEK-293-transfected cells using whole-cell patch-clamp recording. Eleven candidates displayed dose-dependent inhibition of channel currents; among these, NC30 possessing a 4-((1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)piperidin-4-ol heterocyclic core was the most potent one with an IC50 of 4.7μm. These results form a rational basis to design future drugs, and this is the first report of novel TREK1 antagonists delineated by a synergistic application of structure- and ligand-based approaches.

DYN-Mediated Internalization of KCNQ1/KCNE1 Channels under Sustained CPKC Activation

Chronic stimulation of PKC signaling pathways is a hallmark of cardiac disease. Although QT prolongation and arrhythmias have been associated with cardiac disease, the mechanism underlying this prolongation has not been fully understood. Using a combination of time-lapse confocal microscopy and patch-clamp techniques we investigated the time-dependent changes in the subcellular localization and channel activity of cardiac KCNQ1/KCNE1 in response to the prolonged calcium dependent protein kinase C (cPKC) activation. Plasma membrane localization of KCNQ1/KCNE1 was reduced after 30-min cPKC activation, accompanied by decrease in channel conductance. In addition, after the cPKC activation, KCNQ1/KCNE1 channel showed co-localization with the early-endosome marker Rab5. Dephospho-memetric mutation of a putative phosphorylation site in KCEN1 (S102) abolished the channel internalization and current inhibition. Internalization was also inhibited by cPKC-specific inhibitors or co-expression of dominant negative dynamin (DYN-K44A). Finally, inhibition of Rab5 prenylation by statins prevented cPKC-mediated channel internalization. Manipulations that inhibit channel internalization did not affect channel function in the absence of cPKC stimulation. In summary, our results show that Dyn and Rab5 GTPases are key regulators for cell surface expression and internalization of KCNQ1/KCNE1 channels to early endosomes under the chronic cPKC activation. Our findings also indicate that statin treatment may prevent cardiac electrophysiological remodeling under the pathophysiological condition by upregulating the expression levels of KCNQ1/KCNE1 at the plasma membrane in the cardiomyocytes.

Mechanisms underlying atrial-selective block of sodium channels by Wenxin Keli: Experimental and theoretical analysis

IntroductionAtrial-selective inhibition of cardiac sodium channel current (INa) and INa-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study was designed to examine the basis for the atrial-selective actions of Wenxin Keli. MethodsWhole cell INa was recorded at room temperature in canine atrial and ventricular myocytes. Trains of 40 pulses were elicited over a range of pulse durations and interpulse intervals to determine tonic and use-dependent block. A Markovian model for INa that incorporates interaction of Wenxin Keli with different states of the channel was developed to examine the basis for atrial selectivity of the drug. ResultsOur data indicate that Wenxin Keli does not bind significantly to either closed or open states of the sodium channel, but binds very rapidly to the inactivated state of the channel and dissociates rapidly from the closed state. Action potentials recorded from atrial and ventricular preparations in the presence of 5g/L Wenxin Keli were introduced into the computer model in current clamp mode to simulate the effects on maximum upstroke velocity (Vmax). The model predicted much greater inhibition of Vmax in atrial vs. ventricular cells at rapid stimulation rates. ConclusionOur findings suggest that atrial selectivity of Wenxin Keli to block INa is due to more negative steady-state inactivation, less negative resting membrane potential, and shorter diastolic intervals in atrial vs. ventricular cells at rapid activation rates. These actions of Wenxin Keli account for its relatively safe and effective suppression of atrial fibrillation.