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Background: Cardiovascular disease remains one of the leading causes of death globally. Myocardial ischemia and infarction, in particular, frequently cause disturbances in cardiac electrical activity that can trigger ventricular arrhythmias. We aimed to investigate whether catestatin, an endogenous catecholamine-inhibiting peptide, ameliorates myocardial ischemia-induced ventricular arrhythmias in rats and the underlying ionic mechanisms., Methods and Results: Adult male Sprague-Dawley rats were randomly divided into control and catestatin groups. Ventricular arrhythmias were induced by ligation of the left anterior descending coronary artery and electrical stimulation. Action potential, transient outward potassium current, delayed rectifier potassium current, inward rectifying potassium current, and L-type calcium current ( I Ca-L ) of rat ventricular myocytes were recorded using a patch-clamp technique. Catestatin notably reduced ventricular arrhythmia caused by myocardial ischemia/reperfusion and electrical stimulation of rats. In ventricular myocytes, catestatin markedly shortened the action potential duration of ventricular myocytes, which was counteracted by potassium channel antagonists TEACl and 4-AP, and I Ca-L current channel agonist Bay K8644. In addition, catestatin significantly increased transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current density in a concentration-dependent manner. Catestatin accelerated the activation and decelerated the inactivation of the transient outward potassium current channel. Furthermore, catestatin decreased I Ca-L current density in a concentration-dependent manner. Catestatin also accelerated the inactivation of the I Ca-L channel and slowed down the recovery of I Ca-L from inactivation., Conclusions: Catestatin enhances the activity of transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current, while suppressing the I Ca-L in ventricular myocytes, leading to shortened action potential duration and ultimately reducing the ventricular arrhythmia in rats.

Since deuterium replacement has a potential to modulate pharmacodynamics, pharmacokinetics and toxicity, we developed deuterated dronedarone; poyendarone, and assessed its cardiovascular effects. Poyendarone hydrochloride in doses of 0.3 and 3 mg/kg over 30 s was intravenously administered to the halothane-anesthetized dogs (n = 4), which provided peak plasma concentrations of 108 ± 10 and 1120 ± 285 ng/mL, respectively. The 0.3 mg/kg shortened the ventricular repolarization period. The 3 mg/kg transiently increased the heart rate at 5 min but decreased at 45 min, and elevated the total peripheral vascular resistance and left ventricular preload, whereas it reduced the mean blood pressure at 5 min, left ventricular contractility and cardiac output. The transient tachycardic action is considered to be induced by the hypotension-induced, reflex-mediated increase of sympathetic tone. The 3 mg/kg delayed both intra-atrial and intra-ventricular conductions, indicating Na + channel inhibitory action. Moreover, the 3 mg/kg transiently shortened the ventricular repolarization period at 5 min. No significant change was detected in the late repolarization by poyendarone, indicating it might not hardly significantly alter rapidly activating delayed-rectifier K + current (I Kr ). Poyendarone prolonged the atrial effective refractory period greater than the ventricular parameter. When compared with dronedarone, poyendarone showed similar pharmacokinetics of dronedarone, but reduced β-adrenoceptor blocking activity as well as the cardio-suppressive effect. Poyendarone failed to inhibit I Kr and showed higher atrial selectivity in prolonging the effective refractory period of atrium versus ventricle. Thus, the deuteration may be an effective way to improve the cardiovascular profile of dronedarone. Poyendarone is a promising anti-atrial fibrillatory drug candidate.

Background: Rapid delayed rectifier K + current (I Kr ) and late Na + current (I NaL ) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death., Methods: Physiological self AP-clamp was used to measure I NaL and I Kr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between I Kr and I NaL affects repolarization stability in health and disease conditions., Results: We found comparable amount of net charge carried by I Kr and I NaL during the physiological AP, suggesting that outward K + current via I Kr and inward Na + current via I NaL are in balance during physiological repolarization. Remarkably, I Kr and I NaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block I Kr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na + channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit I NaL sufficiently restored APD to control in both cases. Importantly, I NaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, I NaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by I NaL inhibition, increased by I Kr inhibition, and restored by combined I NaL and I Kr inhibitions., Conclusions: Our data demonstrate that I Kr and I NaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.

Background and Purpose: Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs., Experimental Approach: The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes., Key Results: A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide., Conclusions and Implications: Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms., (© 2017 The British Pharmacological Society.)

Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery (RA), and ion channel dysfunction in RA myocytes (RAMs) may be involved in the higher renal vascular resistance. We examined the effects of several voltage-gated K + (K V ) channel blockers on the resting tension in endothelium-denuded RA strips and delayed-rectifier K + currents in RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) fed with low- (Dahl-LS) and high-salt diets (Dahl-HS). The tetraethylammonium (TEA)-induced contraction in RA strips were significantly larger in Dahl-HS than Dahl-LS. Correspondingly, TEA-sensitive K V currents were significantly larger in the RAMs of Dahl-HS than Dahl-LS. Among the TEA-sensitive K V channel subtypes, the expression levels of K V 2.1 transcript and protein were significantly higher in the RA of Dahl-HS than Dahl-LS, while those of K V 1.5, K V 7.1, and K V 7.4 transcripts was comparable in two groups. K V 2.1 currents detected as the guangxitoxin-1E-sensitive component were larger in the RAMs of Dahl-HS than Dahl-LS. These suggest that the up-regulation of the K V 2.1 channel in RAMs may be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.

Introduction: Preclinical in vivo QT measurement as a proarrhythmia essay is expensive and not reliable enough. The aim of the present study was to develop a sensitive, cost-effective, Langendorff perfused guinea pig heart model for proarrhythmia safety screening., Methods: Low concentrations of dofetilide and cisapride (inhibitors of the rapid delayed rectifier potassium current, IKr) were tested alone and co-perfused with HMR-1556 (inhibitor of the slow delayed rectifier potassium current, IKs) in Langendorff perfused guinea pig hearts. The electrocardiographic rate corrected QT (QTc) interval, the Tpeak-Tend interval and the beat-to-beat variability and instability (BVI) of the QT interval were determined in sinus rhythm., Results: Dofetilide and HMR-1556 alone or co-perfused, prolonged the QTc interval by 20±2%, 10±1% and 55±10%, respectively. Similarly, cisapride and HMR-1556 alone or co-perfused, prolonged the QTc interval by 11±3%, 11±4% and 38±6%, respectively. Catecholamine-induced fast heart rate abolished the QTc prolonging effects of the IKr inhibitors, but augmented the QTc prolongation during IKs inhibition. None of the drug perfusions increased significantly the Tpeak-Tend interval and the sinus BVI of the QT interval., Discussion: IKs inhibition increased the QTc prolonging effect of IKr inhibitors in a super-additive (synergistic) manner, and the QTc interval was superior to other proarrhythmia biomarkers measured in sinus rhythm in isolated guinea pig hearts. The effect of catecholamines on the QTc facilitated differentiation between IKr and IKs inhibitors. Thus, QTc measurement in Langendorff perfused guinea pig hearts with pharmacologically attenuated repolarization reserve and periodic catecholamine perfusion seems to be suitable for preclinical proarrhythmia screening., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human cardiomyocytes express 3 distinct types of delayed rectifier potassium channels. Human ether-a-go-go-related gene (hERG) channels conduct the rapidly activating current IKr; KCNQ1/KCNE1 channels conduct the slowly activating current IKs; and Kv1.5 channels conduct an ultrarapid activating current IKur. Here the authors provide a general overview of the mechanistic and structural basis of ion selectivity, gating, and pharmacology of the 3 types of cardiac delayed rectifier potassium ion channels. Most blockers bind to S6 residues that line the central cavity of the channel, whereas activators interact with the channel at 4 symmetric binding sites outside the cavity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A new class III antiarrhythmic drug niferidil has been recently introduced as a highly effective therapy cure for cases of persistent atrial fibrillation, but ionic mechanisms of its action are still unknown. Effects of niferidil on action potential (AP) waveform and major ionic currents were studied in mouse ventricular myocardium. APs were recorded with glass microelectrodes in multicellular preparations of right ventricular wall. Whole-cell patch-clamp technique was used to measure K(+), Ca(2+), and Na(+) currents in isolated mouse ventricular myocytes. While 10(-7) M niferidil failed to alter the AP configuration, 10(-6) M tended to prolong APs (by 12.05 ± 1.8% at 50% of repolarization) and 10(-5) M induced significant slowing of repolarization (32.1 ± 4.9% at 50% of repolarization). Among the potassium currents responsible for AP repolarization phase, IK1 was found to be almost insensitive to niferidil. Ito demonstrated low sensitivity to niferidil with IC50 = 2.03 × 10(-4) M. IKur, which was previously hypothesized to be the main target of the drug, was more sensitive with IC50 = 6 × 10(-5) M. However, sustained delayed rectifier potassium current Iss was inhibited with even lower IC50 = 2.8 × 10(-5) M. Therefore, suppression of Iss and, second, IKur by niferidil seems to underlie the AP prolongation in mouse ventricular tissue. Niferidil also produced a modest decrease in ICaL peak amplitude (IC50≈10(-4) M), but failed to alter INa significantly. Niferidil prolongs APs in mouse ventricular myocardium mainly by inhibiting Iss and IKur K(+) currents, but not exclusively IKur, as was proposed earlier. Further investigations are required to reveal the mechanisms of niferidil action in human myocardium, where IKr is strongly expressed instead of Iss.

Background: Lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and these channels play crucial roles in the lymphocyte activation and proliferation. Since diltiazem and verapamil, which are highly lipophilic Ca(2+) channel blockers (CCBs), exert relatively stronger immunomodulatory effects than the other types of CCBs, they would affect the Kv1.3-channel currents in lymphocytes., Methods: Employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of these drugs on the channel currents and the membrane capacitance., Results: Both diltiazem and verapamil significantly suppressed the peak and the pulse-end currents of the channels, although the effects of verapamil were more marked than those of diltiazem. Both drugs significantly lowered the membrane capacitance, indicating the interactions between the drugs and the plasma membranes., Conclusions: This study demonstrated for the first time that CCBs, such as diltiazem and verapamil, exert inhibitory effects on Kv1.3-channels expressed in lymphocytes. The effects of these drugs may be associated with the mechanisms of immunomodulation by which they decrease the production of inflammatory cytokines., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Objective: To study the effect of allitridum on rapidly delayed rectifier potassium current (IKr) in HEK293 cell line., Methods: HEK293 cells were transiently transfected with HERG channel cDNA plasmid pcDNA3.1 via Lipofectamine. Allitridum was added to the extracellular solution by partial perfusion after giga seal at the final concentration of 30 µmol/L. Whole-cell patch clamp technique was used to record the HERG currents and gating kinetics before and after allitridum exposure at room temperature., Results: The amplitude and density of IHERG were both suppressed by allitridum in a voltage-dependent manner. In the presence of allitridum, the peak current of IHERG was reduced from 73.5∓4.3 pA/pF to 42.1∓3.6 pA/pF at the test potential of +50 mV (P<0.01). Allitridum also concentration-dependently decreased the density of the IHERG. The IC50 of allitridum was 34.74 µmol/L with a Hill coefficient of 1.01. Allitridum at 30 µmol/L caused a significant positive shift of the steady-state activation curve of IHERG and a markedly negative shift of the steady-state inactivation of IHERG, and significantly shortened the slow time constants of IHERG deactivation., Conclusion: Allitridum can potently block IHERG in HEK293 cells, which might be the electrophysiological basis for its anti-arrhythmic action.

Background: The antiarrhythmic potential of a novel multichannel blocker CPUY102122 (CY22) was investigated in the present study., Methods: The effect of CY22 on rapid delayed rectifier potassium channel current (IKr) was studied using whole-cell patch clamp techniques in Chinese Hamster Ovary cells stably expressing human Ether-à-go-go-Related Gene. We further evaluated the antioxidant effects of CY22 and demonstrated the reversal of connexin down-regulation in the development of cardiac ventricular arrhythmias, which was produced using coronary ligation/reperfusion in rabbits. CY22 and Amiodarone were administered 30min prior to the procedure. Next, electrocardiograms were recorded, protein expression of left ventricular Connexin43 (Cx43), non-phosphorylation-Cx43 (np-Cx43), Rac-1 and gp-91[phox] were assayed using Western blot analysis, microstructural changes in the myocardium were observed and redox system activity was assayed., Results: CY22 inhibited IKr in a concentration-dependent manner with IC50 value of 2.8±0.8μmol/L. CY22 treatment significantly decreased T-wave amplitude and QTc arrhythmic scores and ameliorated the shape of the infarcted myocardium compared to the model group. CY22 decreased the serum levels of creatine kinase, lactate dehydrogenase, and myocardial levels of malondialdehyde, as well as increased superoxide dismutase activity. Cx43 expression in the left ventricle was significantly increased by CY22 treatment, which significantly decreased np-43 expression, Rac-1 activity and gp-91[phox] protein expression., Conclusions: These results indicated that CY22 has both antiarrhythmic and cardiovascular protective effects partly by blocking IKr, the production of antioxidants and protection of Cx43., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Dynamics of repolarization, quantified as restitution and electrical memory, impact conduction stability. Relatively less is known about role of slow delayed rectifying potassium current, I(Ks), in dynamics of repolarization and memory compared to the rapidly activating current I(Kr). Trans-membrane potentials were recorded from right ventricular tissues from pigs during reduction (chromanol 293B) and increases in I(Ks) (mefenamic acid). A novel pacing protocol was used to explicitly control diastolic intervals to quantify memory. Restitution hysteresis, a consequence of memory, increased after chromanol 293B (loop thickness and area increased 27 and 38 %) and decreased after mefenamic acid (52 and 53 %). Standard and dynamic restitutions showed an increase in average slope after chromanol 293B and a decrease after mefenamic acid. Increase in slope and memory are hypothesized to have opposite effects on electrical stability; therefore, these results suggest that reduction and enhancement of I(Ks) likely also have offsetting components that affect stability.

Introduction: Escitalopram is rarely associated with prolongation of the QTc interval; however, there are no reported cases of QRS complex widening associated with escitalopram overdose. We report a case of a patient who presented with both QRS complex widening and QTc interval prolongation after an escitalopram overdose., Case: A 16-year-old girl presented to the emergency department after ingestion of escitalopram, tramadol/acetaminophen, and hydrocodone/acetaminophen. Laboratory results were significant for 4-hour acetaminophen 21.1 μg/mL. Serum electrolytes including potassium, magnesium, and calcium were all normal. Initial electrocardiogram (ECG) revealed a widened QRS with an incomplete right bundle branch pattern. After administration of 100-mEq sodium bicarbonate, a repeat ECG revealed narrowing of the QRS complex and a prolonged QTc interval. Magnesium sulfate 2 g intravenous and sodium bicarbonate drip were initiated. A repeat ECG, 1 hour after the second, revealed normalization of the QRS complex and QTc interval., Discussion: Prolongation of the QTc interval is an expected effect of escitalopram. Both escitalopram and citalopram are metabolized to the cardiotoxic metabolite S-didesmethylcitalopram and didesmethylcitalopram, respectively, which have been implicated in numerous cardiac abnormalities including widening of the QRS complex. Although never previously described with escitalopram, this mechanism provides a reasonable explanation for the QRS complex widening and incomplete right bundle branch block that occurred in our patient., Conclusions: Both QRS complex widening and QTc interval prolongation should be monitored in cases of escitalopram and citalopram overdoses.

This study examined the effect of H2O2 on the delayed rectifier potassium current (IKDR) in isolated hippocampal neurons. Whole-cell voltage-clamp experiments were performed on freshly dissociated hippocampal CA1 neurons of SD rats before and after treatment with H2O2. To reveal the mechanism behind H2O2-induced changes in IKDR, cells were treated with different oxidizing and reducing agents. External application of membrane permeable H2O2 reduced the amplitude and voltage-dependence of IKDR in a concentration dependent manner. Desferoxamine (DFO), an iron-chelator that prevents hydroxyl radical (OH) generation, prevented H2O2-induced reduction in IKDR. Application of the sulfhydryl-oxidizing agent 5,5 dithio-bis-nitrobenzoic acid (DTNB) mimicked the effect of H2O2. Sulfhydryl-reducing agents dithiothreitol (DTT) and glutathione (GSH) alone did not affect IKDR; however, DTT and GSH reversed and prevented the H2O2-induced inhibition of IKDR, respectively. Membrane impermeable agents GSH and DTNB showed effects only when added intracellularly identifying intracellular sulfhydryl groups as potential targets for hydroxyl-mediated oxidation. However, the inhibitory effects of DTNB and H2O2 at the positive test potentials were completely and partially abolished by DTT, respectively, suggesting an additional mechanism of action for H2O2, that is not shared by DTNB. In summary, this study provides evidence for the redox modulation of IKDR, identifies hydroxyl radical as an intermediate oxidant responsible for the H2O2-induced decrease in current amplitude and identifies intracellular sulfhydryl groups as an oxidative target., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Inhalation of isoflurane (ISO), a widely used volatile anesthetic, can produce clinical tachypnea. In dogs, this response is reportedly mediated by bronchopulmonary C-fibers (PCFs), but the relevant mechanisms remain unclear. Activation of transient A-type potassium current (IA) channels and delayed rectifier potassium current (IK) channels hyperpolarizes neurons, and inhibition of both channels by ISO increases neural firing. Due to the presence of these channels in the cell bodies of rat PCFs, we determined whether ISO could stimulate PCFs to produce tachypnea in anesthetized rats, and, if so, whether this response resulted from ISO-induced depolarization of the pulmonary C neurons via the inhibition of IA and IK. We recorded ventilatory responses to 5% ISO exposure in anesthetized rats before and after blocking PCF conduction and the responses of pulmonary C neurons (extracellularly recorded) to ISO exposure. ISO-induced (1mM) changes in pulmonary C neuron membrane potential and IA/IK were tested using the perforated patch clamp technique. We found that: (1) ISO inhalation evoked a brief tachypnea (∼7s) and that this response disappeared after blocking PCF conduction; (2) the ISO significantly elevated (by 138%) the firing rate of most pulmonary C neurons (17 out of 21) in the nodose ganglion; and (3) ISO perfusion depolarized the pulmonary C neurons in the vitro and inhibited both IA and IK, and this evoked-depolarization was largely diminished after blocking both IA and IK. Our results suggest that ISO is able to stimulate PCFs to elicit tachypnea in rats, at least partly, via inhibiting IA and IK, thereby depolarizing the pulmonary C neurons., (Copyright © 2013. Published by Elsevier B.V.)

To investigate the electrophysiology mechanisms of new anxiolytic and antidepressant drug: 4-butyl-alpha-agarofuran (AF-5), patch clamp-recording was used to test the effects of AF-5 on voltage-dependent sodium currents, voltage-dependent potassium currents, L-type voltage-dependent calcium currents and GABA dependent Cl(-) currents in primary cultured rat cortical neurons. Effects of AF-5 on Kv2.1 currents, expressed stably in HEK293 cells, were also tested. Our results showed that, delayed rectifier potassium currents (I(K(DR, L-type voltage-dependent calcium currents (I(LC-ca)) in primary cultured rat cortical neurons and Kv2.1 currents in HEK293 cells were significantly inhibited by AF-5, with IC50 as 6.17, 4.4 and 5.29 micromol x L(-1) respectively. However, voltage-dependent sodium currents (I(Na)), GABA dependent Cl(-) currents and transient outward potassium currents (I(K(A)) in primary cultured rat cortical neurons were not significantly blocked by AF-5. Our results concluded that, blocked I(K(DR)) and I(L-Ca) currents may be one of the mechanisms of anxiolytic and antidepression actions of AF-5.

Background: Identified as being the primary mechanism involved in the induction of torsades de pointes (TdP), early after-depolarisation (EAD) formation is an important parameter in cardiac safety pharmacology. Easily observed experimentally at the cellular or tissue level, EAD can also be simulated by computer algorithms using animal or human models. During the last decade, confidence in these algorithms has greatly increased. We investigated the putative usefulness of EAD simulation for cardiac safety pharmacology., Methods: EAD simulations were performed in non-failing human ventricular myocytes using the O'Hara-Rudy dynamic model. The role of each cardiac current was investigated by modifying the amplitude of its activity in the model. Prediction of EAD induction by drugs was based on the ratio of their 50% inhibitory concentration values for various cardiac ionic currents to their maximal effective free therapeutic plasma concentration (EFTPCmax)., Results: In the ventricular endocardial myocytes, EAD was only induced by at least 85% inhibition of the rapid delayed rectifier K(+) current (IKr). The other currents can either induce or prevent EAD under sub- (80% IKr inhibition) or up-threshold conditions (87% IKr inhibition) of EAD. The study of the ability of drugs to induce EAD resulted in a classification which was in agreement with the Tdp risk classification., Conclusion: Based on EAD computer simulation within the human situation, the present study identified the role of various cardiac currents in the EAD formation and suggested that prediction of EAD formation can be useful for early cardiac safety pharmacology.

Background: We evaluated the viability of I(Kur) as a target for maintenance of sinus rhythm in patients with a history of atrial fibrillation through the testing of MK-0448, a novel I(Kur) inhibitor., Methods and Results: In vitro MK-0448 studies demonstrated strong inhibition of I(Kur) with minimal off-target activity. In vivo MK-0448 studies in normal anesthetized dogs demonstrated significant prolongation of the atrial refractory period compared with vehicle controls without affecting the ventricular refractory period. In studies of a conscious dog heart failure model, sustained atrial fibrillation was terminated with bolus intravenous MK-0448 doses of 0.03 and 0.1 mg/kg. These data led to a 2-part first-in-human study: Part I evaluated safety and pharmacokinetics, and part II was an invasive electrophysiological study in healthy subjects. MK-0448 was well-tolerated with mild adverse experiences, most commonly irritation at the injection site. During the electrophysiological study, ascending doses of MK-0448 were administered, but no increases in atrial or ventricular refractoriness were detected, despite achieving plasma concentrations in excess of 2 μmol/L. Follow-up studies in normal anesthetized dogs designed to assess the influence of autonomic tone demonstrated that prolongation of atrial refractoriness with MK-0448 was markedly attenuated in the presence of vagal nerve simulation, suggesting that the effects of I(Kur) blockade on atrial repolarization may be negated by enhanced parasympathetic neural tone., Conclusions: The contribution of I(Kur) to human atrial electrophysiology is less prominent than in preclinical models and therefore is likely to be of limited therapeutic value for the prevention of atrial fibrillation.

Polychlorinated biphenyls (PCBs) have been known as serious persistent organic pollutants (POPs), causing developmental delays and motor dysfunction. We have investigated the effects of two PCB congeners, 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on ECG, action potential, and the rapidly activating delayed rectifier K+ current (I(Kr)) of guinea pigs' hearts, and hERG K+ current expressed in Xenopus oocytes. PCB 126 shortened the corrected QT interval (QTc) of ECG and decreased the action potential duration at 90% (APD(90)), and 50% of repolarization (APD₅₀) (P<0.05) without changing the action potential duration at 20% (APD₂₀). PCB 77 decreased APD₂₀ (P<0.05) without affecting QTc, APD₉₀, and APD₅₀. The PCB 126 increased the I(Kr) in guinea-pig ventricular myocytes held at 36°C and hERG K+ current amplitude at the end of the voltage steps in voltage-dependent mode (P<0.05); however, PCB 77 did not change the hERG K+ current amplitude. The PCB 77 increased the diastolic Ca²⁺ and decreased Ca²⁺ transient amplitude (P<0.05), however PCB 126 did not change. The results suggest that PCB 126 shortened the QTc and decreased the APD₉₀ possibly by increasing I(Kr), while PCB 77 decreased the APD₂₀ possibly by other modulation related with intracellular Ca²⁺. The present data indicate that the environmental toxicants, PCBs, can acutely affect cardiac electrophysiology including ECG, action potential, intracellular Ca²⁺, and channel activity, resulting in toxic effects on the cardiac function in view of the possible accumulation of the PCBs in human body., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rationale: Eugenol (EUG, 4-allyl-2-methoxyphenol), the main component of essential oil extracted from cloves, has various uses in medicine because of its potential to modulate neuronal excitability. However, its effects on the ionic mechanisms remains incompletely understood., Objectives: We aimed to investigate EUG's effects on neuronal ionic currents and excitability, especially on voltage-gated ion currents, and to verify the effects on a hyperexcitability-temporal lobe seizure model., Methods: With the aid of patch-clamp technology, we first investigated the effects of EUG on ionic currents in NG108-15 neuronal cells differentiated with cyclic AMP. We then used modified Pinsky-Rinzel simulation modeling to evaluate its effects on spontaneous action potentials (APs). Finally, we investigated its effects on pilocarpine-induced seizures in rats., Results: EUG depressed the transient and late components of I(Na) in the neurons. It not only increased the degree of I(Na) inactivation, but specifically suppressed the non-inactivating I(Na) (I(Na(NI))). Its inhibition of I (Na(NI)) was reversed by tefluthrin. In addition, EUG diminished L-type Ca(2+) current and delayed rectifier K(+) current only at higher concentrations. EUG's effects on APs frequency reduction was verified by the simulation modeling. In pilocarpine-induced seizures, the EUG-treated rats showed no shorter seizure latency but a lower seizure severity and mortality than the control rats. The EUG's effect on seizure severity was occluded by the I(Na(NI)) antagonist riluzole., Conclusion: The synergistic blocking effects of I (Na) and I(Na(NI)) contributes to the main mechanism through which EUG affects the firing of neuronal APs and modulate neuronal hyperexcitability such as pilocarpine-induced temporal lobe seizures.

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K(+)) current I(Kr). Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K(+) currents I(Kr) and I(Ks), the L-type calcium ion (Ca(2+)) current I(Ca,L), and the peak sodium ion (Na(+)) current I(Na) and increased the persistent Na(+) current I(NaP). Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in I(NaP). These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

Photochromic channel blockers provide a conceptually simple and convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of K(v) channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.

Ectothermic vertebrates experience acute and chronic temperature changes which affect cardiac excitability and may threaten electrical stability of the heart. Nevertheless, ectothermic hearts function over wide range of temperatures without cardiac arrhythmias, probably due to special molecular adaptations. We examine function and molecular basis of the slow delayed rectifier K(+) current (I(Ks)) in cardiac myocytes of a eurythermic fish (Carassius carassius L.). I(Ks) is an important repolarizing current that prevents excessive prolongation of cardiac action potential, but it is extremely slowly activating when expressed in typical molecular composition of the endothermic animals. Comparison of the I(Ks) of the crucian carp atrial myocytes with the currents produced by homomeric K(v)7.1 and heteromeric K(v)7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the I(Ks) are similar to those of the homomeric K(v)7.1 channels. Consistently with electrophysiological properties and homomeric K(v)7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6-1.9% of the expression level of the K(v)7.1 subunit. Since activation kinetics of the homomeric K(v)7.1 channels is much faster than activation of the heteromeric K(v)7.1/MinK channels, the homomeric K(v)7.1 composition of the crucian carp cardiac I(Ks) is thermally adaptive: the slow delayed rectifier channels can open despite low body temperatures and curtail the duration of cardiac action potential in ectothermic crucian carp. We suggest that the homomeric K(v)7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric K(v)7.1/MinK channels evolved later to adapt I(Ks) to high body temperature of endotherms.

Background: Early development of cardiac hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents can be modulated by the activation of humoral factors, such as angiotensin II (ANG II) through protein kinases. The aim of this work is to assess the regulation of IK and IK1 by ANG II through the PI3-K pathway in hypertrophied ventricular myocytes., Material/methods: Cardiac eccentric hypertrophy was induced through volume-overload in adult male rats by aorto-caval shunt (3 weeks). After one week half of the rats were given captopril (2 weeks; 0.5 g/l/day) and the other half served as control. The voltage-clamp and western blot techniques were used to measure the delayed outward rectifier potassium current (IK) and the instantaneous inward rectifier potassium current (IK1) and Akt activity, respectively., Results: Hypertrophied cardiomyocytes showed reduction in IK and IK1. Treatment with captopril alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10-6M) to cardiocytes treated with captopril reduced IK and IK1 in shunts, but not in sham. Captopril treatment reversed ANG II effects on IK and IK1 in a PI3-K-independent manner. However in the absence of angiotensin converting enzyme inhibition, ANG II increased both IK and IK1 in a PI3-K-dependent manner in hypertrophied cardiomyocytes., Conclusions: Thus, captopril treatment reveals a negative effect of ANG II on IK and IK1, which is PI3-K independent, whereas in the absence of angiotensin converting enzyme inhibition IK and IK1 regulation is dependent upon PI3-K.

Objective: Ginkgo biloba extract 50 (GBE50) is a new multicomponent drug with a polyvalent action extracted from the leave of Ginkgo biloba. The aim of this experiment was to study the effects of GBE50 on delayed rectifier potassium current (I(K)) in ventricular myocytes under normal and simulated ischemia conditions in guinea pigs., Methods: Single ventricular myocytes were isolated by an enzymatic dissociation method. I(K) were recorded by whole-cell patch clamp technique in voltage clamp mode. GBE50 was added to the perfusion chamber from low to high concentrations (25, 50,100 mg/L) in normal condition. Different concentrations of GBE50 (25, 50, 100 mg/L) were prepared with simulated ischemic fluid., Results: (1) Under normal condition, 100 mg/L GBE50 decreased I(K) (n = 7, P < 0.05). (2) Under ischemia condition, it was observed that I(K) was inhibited (n = 8, P < 0.05). (3) Perfusion with ischemia solution containing 50 mg/L (n = 8, P > 0.05) and 100 mg/L GBE50 (n = 6, P > 0.05) could reverse the decrease of I(K)., Conclusion: GBE50 significantly decreased I(K) in a concentration-dependent manner. GBE50 could alleviate the electrophysiological heterogeneity of myocardium to prevent ischemic myocardium from arrhythmia.

Background: Previous studies demonstrated general anesthetics affect potassium ion channels, which may be one of the mechanisms of general anesthesia. Because the effect of etomidate on potassium channels in rat hippocampus which is involved in memory function has not been studied, we investigated the effects of etomidate on both delayed rectifier potassium current (I(K(DR))) and transient outward potassium current (I(K(A))) in acutely dissociated rat hippocampal pyramidal neurons., Methods: Single rat hippocampal pyramidal neurons from male Wistar rats of - 10 days were acutely dissociated by enzymatic digestion and mechanical dispersion according to the methods of Kay and Wong with slight modification. Voltage-clamp recordings were performed in the whole-cell patch clamp configuration. Currents were recorded with a List EPC-10 amplifier and data were stored in a computer using Pulse 8.5. Student's paired two-tail t test was used for data analysis., Results: At the concentration of 100 micromol/L, etomidate significantly inhibited I(K(DR)) by 49.2% at +40 mV when depolarized from -110 mV (P < 0.01, n = 8), while did not affect I(K(A)) (n = 8, P > 0.05). The IC(50) value of etomidate for blocking I(K(DR)) was calculated as 5.4 micromol/L, with a Hill slope of 2.45. At the presence of 10 micromol/L etomidate, the V1/2 of activation curve was shifted from (17.3 +/- 1.5) mV to (10.7 +/- 2.9) mV (n = 8, P < 0.05), the V1/2 of inactivation curve was shifted from (-18.3 +/- 2.2) mV to (-45.3 +/- 9.4) mV (n = 8, P < 0.05). Etomidate 10 micromol/L shifted both the activation curve and inactivation curve of I(K(DR)) to negative potential, but mainly affected the inactivation kinetics., Conclusions: Etomidate potently inhibited I(K(DR)) but not I(K(A)) in rat hippocampal pyramidal neurons. I(K(DR)) was inhibited by etomidate in a concentration-dependent manner, while I(K(A)) remained unaffected.

Ion channels are known to be modulated by antidepressant drugs, but the molecular mechanisms are not known. We have shown that the antidepressant drug amoxapine suppresses rectifier outward K(+) (I(K)) currents in mouse cortical neurons. At a concentration of 10 to 500 muM, amoxapine reversibly inhibited I(K) in a dose-dependent manner and modulated both steady-state activation and inactivation properties. The application of forskolin or dibutyryl cAMP mimicked the inhibitory effect of amoxapine on I(K) and abolished further inhibition by amoxapine. N-[2-(p-Bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide (H-89), a protein kinase A (PKA) inhibitor, augmented I(K) amplitudes and completely eliminated amoxapine inhibition of I(K). Amoxapine was also found to significantly increase intracellular cAMP levels. The effects of amoxapine on I(K) were abolished by preincubation with 5-hydroxytryptamine (5-HT) and the antagonists of 5-HT(2) receptor. Moreover, intracellular application of guanosine 5'-[gammathio]-triphosphate increased I(K) amplitudes and prevented amoxapine-induced inhibition. The selective Kv2.1 subunit blocker Jingzhaotoxin-III reduced I(K) amplitudes by 30% and also significantly abolished the inhibitory effect of amoxapine. Together these results suggest that amoxapine inhibits I(K) in mouse cortical neurons by cAMP/PKA-dependent pathway associated with the 5-HT receptor, and suggest that the Kv2.1 alpha-subunit may be the target for this inhibition.

Cardiac K(+) channels are cardiomyocyte membrane proteins that regulate K(+) ion flow across the cell membrane on the electrochemical gradient and determine the resting membrane potential and the cardiac action potential morphology and duration. Several K(+) channels have been well studied in the human heart. They include the transient outward K(+) current I(to1), the ultra-rapidly activating delayed rectifier current I(Kur), the rapidly and slowly activating delayed rectifier currents I(Kr) and I(Ks), the inward rectifier K(+) current I(K1), and ligand-gated K(+) channels, including adenosine-5'-triphosphate (ATP)-sensitive K(+) current (I(KATP)) and acetylcholine-activated current (I(KACh)). Regional differences of K(+) channel expression contribute to the variable morphologies and durations of cardiac action potentials from sinus node and atrial to ventricular myocytes, and different ventricular layers from endocardium and midmyocardium to epicardium. They also show different responses to endogenous regulators and/or pharmacological agents. K(+) channels are well-known targets for developing novel anti-arrhythmic drugs that can effectively prevent/inhibit cardiac arrhythmias. Especially, atrial-specific K(+) channel currents (I(Kur) and I(KACh)) are the targets for developing atrial-selective anti-atrial fibrillation drugs, which has been greatly progressed in recent years. This chapter concentrates on recent advances in intracellular signaling regulation and pharmacology of cardiac K(+) channels under physiological and pathophysiological conditions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: Doxorubicin, an anthracycline widely used in the treatment of a broad range of tumours, causes acute QT prolongation. Dexrazoxane has been shown to prevent the QT prolongation induced by another anthracycline, epirubicin, but has not yet been reported to prevent that induced by doxorubicin. Thus, the present study was designed to test whether the acute QT effects induced by doxorubicin could be blocked by dexrazoxane and to explore the mechanism. Results were compared with those obtained with a reference human ether-a-go-go (hERG) channel blocker, moxifloxacin., Methods: The effects of moxifloxacin (100 microM) and doxorubicin (30 microM), with or without dexrazoxane (from 3 to 30 microM), have been evaluated on the QTc interval in guinea-pig isolated hearts and on I(Kr) (rapid component of the delayed rectifier current) and I(Ks) (slow component of the delayed rectifier current) currents stably expressed in human embryonic kidney 293 cells., Results: Moxifloxacin (100 microM), a potent hERG blocker, prolonged QTc by 22%, and this effect was not prevented by dexrazoxane. Doxorubicin (30 microM) also prolonged QTc by 13%, did not significantly block hERG channels and specifically inhibited I(Ks) (IC(50): 4.78 microM). Dexrazoxane significantly reduced the doxorubicin-induced QTc prolongation and prevented doxorubicin-induced inhibition of I(Ks)., Conclusion and Implications: Doxorubicin acutely prolonged the QT interval in guinea-pig heart by selective I(Ks) blockade. This effect was prevented by dexrazoxane. This result is important because it illustrates the danger of neglecting I(Ks) in favour of hERG screening alone, for early preclinical testing for possible induction of torsade de pointes.

The ionic current responsible for terminating the action potential (AP), and thereby in part determining the AP duration (APD), is the potassium current (IK), consisting of primarily two components: a rapidly (IKr) and a slowly (IKs) activating delayed rectifier potassium current. The aim of this study was to evaluate potential antiarrhythmic effects of compound induced IKs activation using the benzodiazepine L-364,373 (R-L3). Ventricular myocytes from guinea pigs were isolated and whole-cell current clamping was performed at 35 degrees C. It was found that 1 microM R-L3 significantly reduced the APD90 at pacing frequencies of 1, 2, and 4 Hz when compared to control (40 +/- 6%, 22 +/- 2%, and 32 +/- 2%, respectively). The reduction of APD90 was accompanied by a reduced triangulation (given as APD30-90) when compared to control at all pacing frequencies (62 +/- 7 ms vs. 41 +/- 3 ms, 55 +/- 5 ms vs. 35 +/- 6 ms, and 45 +/- 4 ms vs. 32 +/- 2 ms, at 1 Hz, 2 Hz, and 4 Hz, respectively). The abbreviated APDs also resulted in a reduction in the relative refractory period, and no direct protection against pacing induced early after-depolarizations (EAD) could be observed. However, an increase in repolarizing capacity was seen with 1 microM R-L3, as more complete repolarization of the AP was achieved before EADs could be elicited. Finally, a functional demonstration of the repolarization reserve revealed that increased IKs can counteract a pharmacologically reduced IKr. In conclusion, pharmacological activation of IKs possesses both pro- and antiarrhythmic characters. The most prominent antiarrhythmic propensity is the ability for IKs activation to rescue a cellular model of long QT type 2.

Background: Dexmedetomidine (DEX), a selective agonist of alpha2-adrenergic receptors, is recognized to facilitate analgesia and anaesthesia in humans. Despite the potential for wide use, its effects on ion currents and membrane potential in neurones remain largely unclear., Methods: We investigated the effects of DEX on ion channels in NG108-15 neuronal cells differentiated with dibutyryl cyclic AMP and in cultured cerebellar neurones., Results: DEX suppressed the amplitude of delayed rectifier K+ current [I(K(DR))] in a concentration-dependent manner with an IC50 value of 4.6 microM in NG108-15 cells. No change in the steady-state inactivation of I(K(DR)) was evident in the presence of DEX. A minimal binding scheme was also used to evaluate DEX-induced block of I(K(DR)). Inhibition of I(K(DR)) by DEX was still observed in cells preincubated with yohimbine (10 microM) or efaroxan (10 microM). DEX depressed the peak amplitude of Na+ current (I(Na)), whereas it had minimal effect on L-type Ca2+ current. Under current-clamp configuration, DEX increased the duration of action potentials (APs). I(K(DR)) and I(Na) in response to AP waveforms were more sensitive to block by DEX than those elicited during rectangular pulses. In isolated cerebellar granule cells, DEX also effectively suppressed I(K(DR))., Conclusions: The effects of DEX are not limited to its interactions with alpha2-adrenergic receptors. Inhibitory effects on I(K(DR)) and I(Na) constitute one of the underlying mechanisms through which DEX and its structurally related compounds might affect neuronal activity in vivo.

Propofol is a widely used intravenous general anesthetic. The negative inotropic effect of propofol has been best explained by inhibition of the L-type Ca(2+) current (I(Ca)). Using guinea-pig cardiac preparations, however, we found that the propofol concentration producing a 50% decrease in force of contraction was more than 10 times higher than that producing a 50% inhibition of I(Ca), implying that a compensatory mechanism may be present to counteract the negative inotropic effect associated with the I(Ca) inhibition. Consistent with I(Ca) inhibition, propofol produced a shortening of action potential duration (APD) in single cardiomyocytes. Yet, the concentrations necessary to shorten APD were greater than that for 50% inhibition of I(Ca). This was associated with the potent and effective inhibition of the slowly activating component of the delayed rectifier K(+) current (I(Ks)). Thus, the I(Ks) blockade with propofol may counterbalance the APD shortening evoked by its I(Ca) inhibition. Taken together, the negative inotropic effect of propofol is detectable only at supratherapeutic concentrations. At clinically relevant concentrations, the action potential prolongation mechanism due to I(Ks) inhibition appears to alleviate the reduction in transsarcolemmal Ca(2+) influx through L-type Ca(2+) channels, which may help to counteract the net negative inotropism of propofol.

This study examined the role of the actin cytoskeleton in Rho-kinase-mediated suppression of the delayed-rectifier K(+) (K(DR)) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated, and whole cell K(DR) currents were monitored using conventional patch-clamp electrophysiology. At +40 mV, the K(DR) current averaged 19.8 +/- 1.6 pA/pF (mean +/- SE) and was potently inhibited by UTP (3 x 10(-5) M). This suppression was observed to depend on Rho signaling and was abolished by the Rho-kinase inhibitors H-1152 (3 x 10(-7) M) and Y-27632 (3 x 10(-5) M). Rho-kinase was also found to concomitantly facilitate actin polymerization in response to UTP. We therefore examined whether actin dynamics played a role in the ability of Rho-kinase to suppress K(DR) current and found that actin disruption using either cytochalasin D (1 x 10(-5) M) or latrunculin A (1 x 10(-8) M) prevented current modulation. Consistent with our electrophysiological observations, both Rho-kinase inhibition and actin disruption significantly attenuated UTP-induced depolarization and constriction of cerebral arteries. We propose that UTP initiates Rho-kinase-mediated remodeling of the actin cytoskeleton and consequently suppresses the K(DR) current, thereby facilitating the depolarization and constriction of cerebral arteries.

Aim: To study the effect of Sphingosine-1-phosphate on the delayed rectifier potassium current (IK) and the inward rectifier potassium current (IK1) of guinea pig isolated ventricular myocytes., Methods: Whole cell patch clamp technique was applied to record the delayed rectifier potassium current and the delayed rectifier potassium current of guinea pig isolated ventricular myocytes., Results: (1) IK of S1P (1.1 micromol/L) decreased from (1.2 +/- 0.26)nA to (0.95 +/- 0.23)nA. While IK of S1P (2.2 micromol/L) decreased from (1.43 +/- 0.31)nA to (1.02 +/- 0.28)nA. There was significant difference compared to control group (P < 0.01, n = 6). The IK peak value was decreased from (1.29 +/- 0.26) nA to (1.26 +/- 0.37)nA at the group of S1P (1.1 micromol/L) plus suramin (200 micromol/L) and showed no significant difference compared to control group (P > 0.05, n = 6). (2) IK1 of S1P (1.1 micromol/L) decreased from (-8.94 +/- 2.01)nA to (-8.81 = 1.55)nA. While IK of S1P (2.2 micromol/L) decreased from (-8.86 +/- 1.59)nA to (-8.55 +/- 1.39)nA. There was no significant difference compared to control group (P > 0.05, n = 6)., Conclusion: S1P inhibits delayed rectifier potassium current of ventricular myocyte in guinea pig remarkably, S1P shows no effects on delayed rectifier potassium current of ventricular myocyte in guinea pig.

Caffeine, a prototypic bitter stimulus, produces several physiological actions on taste receptor cells that include inhibition of KIR and KV potassium currents and elevations of intracellular calcium. These responses display adaptation, i.e. their magnitude diminishes in the sustained presence of the stimulus. Levels of the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) are well known to modulate many potassium channels, activating the channel by stabilizing its open state. Here we investigate a putative relationship of KIR and KV with PIP2 levels hypothesizing that inhibition of these currents by caffeine might be allayed by PIP2 resynthesis. Using standard patch-clamp techniques, recordings of either potassium current from rat posterior taste receptor cells produced essentially parallel results when PIP2 levels were manipulated pharmacologically. Increasing PIP2 levels by blocking phosphoinositide-3 kinase with wortmannin or LY294002, or by blocking phospholipase C with U73122 all significantly increased the incidence of adaptation for both KIR and KV. Conversely, lowering PIP2 synthesis by blocking PI4K or using the PIP2 scavengers polylysine or bovine serum albumin reduced the incidence of adaptation. Adaptation could be modulated by activation of protein kinase C but not calcium calmodulin kinase. Collectively, these data support two highly novel conclusions: potassium currents in taste receptor cells are significantly modulated by PIP2 levels and PIP2 resynthesis may play a central role in the gustatory adaptation process at the primary receptor cell level.

Recent studies implicate the possible neurotoxicity of SO(2), however, its mechanisms remain unclear. In the present study, we investigated SO(2) derivative-induced effect on delayed rectifier potassium channels (I(K)) and cellular death/apoptosis in primary cultured hippocampal neurons. The results demonstrate that SO(2) derivatives (NaHSO(3) and Na(2)SO(3), 3:1M/M) effectively augmented I(K) and promoted the activation of delayed rectifier potassium channels. Also, SO(2) derivatives increased neuronal death percentage and contributed to the formation of DNA ladder in concentration-dependent manners. Interestingly, the neuronal death and DNA ladder formation, caused by SO(2) derivatives, could be attenuated by the delayed rectifier potassium channel blocker (tetraethylammonium, TEA), but not by the transient outward potassium channel blocker (4-aminopyridine, 4-AP). It implies that stimulating delayed rectifier potassium channels were involved in SO(2) derivative-caused hippocampal neuronal insults, and blocking these channels might be one of the possibly clinical treatment for SO(2)-caused neuronal dysfunction.

As an acetylcholinesterase (AChE) inhibitor, the effects of 3-benzidino-6-phenylpyridazine (BPP) on outward potassium current including delayed rectifier potassium current (I(K(DR))) and transient outward potassium current (I(K(A))) in acutely isolated rat hippocampal pyramidal neurons were studied, using the whole cell patch-clamp technique. BPP reversibly inhibited electric eel AChE as an inhibitor, with IC(50) of 1.43 microM. BPP (0.10-100 microM) decreased I(K(DR)) and I(K(A)) in a concentration-dependent, voltage-independent and partial reversible manner, with IC(50) of 0.47 and 0.31 microM, respectively. 10 microM BPP did not affect steady-state activation of I(K(DR)) and I(K(A)). In addition, 10 microM BPP shifted the voltage dependence of steady-state inactivation of I(K(A)) towards negative potential. In conclusion, BPP potently inhibits I(K(DR)) and I(K(A)) in rat hippocampal pyramidal neurons, which may contribute to BPP's restoring the damaged central nervous system.

Background: Block of ultrarapid delayed rectified potassium current (I(Kur)), present in atria but not in ventricles, is thought to be a promising approach for atrial-specific therapy of atrial fibrillation (AF). However, it has been shown that I(Kur) block may abbreviate atrial repolarization and that loss-of-function mutations in KCNA5, which encodes K(v) 1.5 channels responsible for I(Kur), is associated with familial AF., Objective: Our objective in this study was to use low concentrations of 4-aminopyridine (4-AP, 10 to 50 microM), known to selectively block I(Kur), to assess the proarrhythmic and antiarrhythmic effects of I(Kur) block in healthy and remodeled atria., Methods: Isolated canine coronary-perfused right atrial preparations were used. Acetylcholine or ischemia/reperfusion was used to acutely remodel the atria. Transmembrane action potentials and a pseudo-electrocardiogram were simultaneously recorded., Results: Normal (healthy) atria typically displayed action potentials (AP) with a prominent plateau, whereas remodeled atria displayed triangular-shaped APs (remodeled). In healthy atria, in which AF could not be induced with programmed stimulation, 4-AP abbreviated action potential measured at 90% repolarization (APD(90)) and effective refractory period (ERP), permitting the induction of AF in 4 of 12 preparations (33%). In remodeled atria, 4-AP produced little (50 microM) to no (10 to 25 microM) prolongation of APD(90) or ERP and was either ineffective or poorly effective in terminating AF or preventing its induction., Conclusion: Our findings suggest that block of I(Kur) can provide the substrate for development of AF in healthy canine atria, presumably via abbreviation of APD and ERP.

DPI 201-107 (DPI), a diphenylpiperazinylindole derivative, was reported to be a cardio-selective modifier of voltage-gated Na+ channels. It remains unclear whether DPI has any effects on ion currents. The effects of DPI on ion currents and membrane potential in pituitary tumor (GH3) cells were investigated in this study. DPI (1-100 microM) suppressed the amplitude of delayed-rectifier K+ current (I(K(DR))) in a concentration-dependent manner with an IC(50) value of 9.4 microM. The presence of DPI also enhanced the rate and extent of I(K(DR)) inactivation. Recovery from block by DPI (10 microM) was fitted by a single exponential. Crossover of tail currents during the exposure to DPI was also observed. Under current-clamp recordings, DPI prolonged action potential duration in GH3 cells. With a minimal binding scheme, DPI-induced block of I(K(DR))) was quantitatively provided. The exposure to DPI also blocked I(K(DR))) with a concomitant increase in current inactivation in NG108-15 neuronal cells. Taken together, the results imply that DPI acts as an open-channel blocker of delayed-rectifier K+ channels in these cells. The widening of action potentials induced by DPI in these cells may be explained mainly by its block of I(K(DR))) in a state-dependent manner.

The properties of slowly inactivating delayed-rectifier K+ current (I(K)(dr)) were investigated in NG108-15 neuronal cells differentiated with long-term exposure to dibutyryl cyclic AMP. Slowly inactivating I(K)(dr) could be elicited by prolonged depolarizations from -50 to +50 mV. These outward K+ currents were found to decay at potentials above -20 mV, and the decay became faster with greater depolarization. Cell exposure to aconitine resulted in the reduction of I(K)(dr) amplitude along with an accelerated decay of current inactivation. Under current-clamp recordings, a delay in the initiation of action potentials (APs) in response to prolonged current stimuli was observed in these cells. Application of aconitine shortened the AP initiation in combination with an increase in both width of spike discharge and firing frequency. The computer model, in which state-dependent inactivation of I(K)(dr) was incorporated, was also implemented to predict the firing behavior present in NG108-15 cells. As the inactivation rate constant of I(K)(dr) was elevated, the firing frequency was progressively increased along with a shortening of the latency for AP appearance. Our theoretical work and the experimental results led us to propose a pivotal role of slowly inactivating I(K)(dr) in delayed firing of APs in NG108-15 cells. The results also suggest that aconitine modulation of I(K)(dr) gating is an important molecular mechanism through which it can contribute to neuronal firing.

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the hyposmotic stimulation of cardiac I Ks, a slowly activating delayed-rectifier K+ current that promotes repolarization of the action potential. The current was recorded from whole-cell-configured guinea-pig ventricular myocytes before, during, and after their exposure to solution whose osmolarity was 0.75 times normal. Exposure to hyposmotic solution caused a near-doubling of the amplitude of I Ks, with little change in the voltage dependence of current activation. Stable, hyposmotically stimulated I Ks (I Ks,Hypo) was decreased by broadspectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC50 approximately 5 microM) and tyrphostin A25 (IC50 15.8 +/- 1.6 microM) but not by TK-inactive tyrphostin analogs, suggesting that tyrosine phosphorylation is important for maintenance of the current. In agreement with that view, we found that the TK-inhibitor action on I Ks,Hypo was strongly antagonized by vanadate compounds known to inhibit phosphotyrosyl phosphatase. When myocytes were pretreated with TK inhibitors, the stimulation of I Ks was attenuated in a concentration-dependent manner. The attenuation was not due to concomitant attenuation of a stimulation of tyrosine phosphorylation because neither the stimulation of I Ks nor its rate of decay following removal of hyposmotic solution was affected by pretreatment with vanadates. We suggest that the stimulation of I Ks by hyposmotic solution is dependent on a basal tyrosine phosphorylation that modulates a swelling-induced I Ks-stimulatory signal and/or the receptivity of Ks channels to that signal.

The effects of cadmium (Cd(2+)) on the transient outward potassium current (I(A)) and delayed rectifier potassium current (I(K)) were investigated in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp technique. The results showed that Cd(2+) inhibited the amplitudes of I(A) and I (K) in a reversible and concentration-dependent manner, with half-maximal inhibitive concentration (IC(50)) values of 546+/-59 and 749+/-53 microM, and the inhibitory effect of Cd(2+) was voltage dependent. Cd(2+) significantly shifted the steady-state activation and inactivation curve of I(A) to more positive potentials. In contrast, Cd(2+) caused a relatively less but still significant positive shift in the activation of I(K) without effect on the inactivation curve. Cd(2+) significantly slowed the recovery from inactivation of I(K) but had no effect on the recovery time course of I(A). The results suggest that the modulation of I(A) and I(K) was most likely mediated by the interaction of Cd(2+) with a specific site on the potassium-channel protein rather than by screening of bulk surface-negative charge. The effects of Cd(2+) on the voltage-gated potassium currents may be a possible contributing mechanism for the Cd(2+)-induced neurotoxic damage. In addition, the effects of Cd(2+) on the potassium currents at concentrations that overlap with its effects on calcium currents raise concerns about its use in pharmacological or physiological studies.

The effects of aconitine (ACO), a highly toxic alkaloid, on ion currents in differentiated NG108-15 neuronal cells were investigated in this study. ACO (0.3-30 microM) suppressed the amplitude of delayed rectifier K+ current (I K(DR)) in a concentration-dependent manner with an IC50 value of 3.1 microM. The presence of ACO enhanced the rate and extent of I K(DR) inactivation, although it had no effect on the initial activation phase of I K(DR). It could shift the inactivation curve of I K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I K(DR) was also enhanced by ACO. Orphenadrine (30 microM) or methyllycaconitine (30 microM) slightly suppressed I K(DR) without modifying current decay. ACO (10 microM) had an inhibitory effect on voltage-dependent Na+ current (I Na). Under current-clamp recordings, ACO increased the firing and widening of action potentials in these cells. With the aid of the minimal binding scheme, the ACO actions on I K(DR) was quantitatively provided with a dissociation constant of 0.6 microM. A modeled cell was designed to duplicate its inhibitory effect on spontaneous pacemaking. ACO also blocked I K(DR) in neuroblastoma SH-SY5Y cells. Taken together, the experimental data and simulations show that ACO can block delayed rectifier K+ channels of neurons in a concentration- and state-dependent manner. Changes in action potentials induced by ACO in neurons in vivo can be explained mainly by its blocking actions on I K(DR) and I Na.

The presence of serum in biological samples often negatively impacts the quality of in vitro assays. However, assays tolerant of serum are useful for assessing the in vivo availability of a small molecule for its target. Electrophysiology assays of ion channels are notoriously sensitive to serum because of their reliance on the interaction of the plasma membrane with a recording electrode. Here we investigate the tolerance of an automated electrophysiology assay for a voltage-gated potassium (K(V)) channel to serum and purified plasma proteins. The delayed rectifier channel, K(V)2.1, stably expressed in Chinese hamster ovary cells produces large, stable currents on the IonWorks Quattro platform (MDS Analytical Technologies, Sunnyvale, CA), making it an ideal test case. K(V)2.1 currents recorded on this platform are highly resistant to serum, allowing recordings in as high as 33% serum. Using a set of compounds related to the K(V) channel blocker, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone, we show that shifts in compound potency with whole serum or isolated serum proteins can be reliably measured with this assay. Importantly, this assay is also relatively insensitive to plasma, allowing the creation of a bioassay for inhibitors of K(V)2.1 channel activity. Here we show that such a bioassay can quantify the levels of the gating modifier, guangxitoxin-1E, in plasma samples from mice dosed with the peptide. This study demonstrates the utility of using an automated electrophysiology platform for measuring serum shifts and for bioassays of ion channel modulators.

The authors previously demonstrated that isoflurane, a widely used volatile anesthetic, induced depolarization and increased the frequency of spontaneous action potentials in principal dopamine neurons in rat substantia nigra pars compacta. We studied the effects of isoflurane on voltage-dependent K channels to clarify the mechanisms of the increase in excitability in these neurons. Voltage-clamp whole-cell recordings were made in rat midbrain slices. We recorded the outward membrane currents in response to depolarizing voltage steps from -120 mV and -25 mV and isolated the transient outward current mediated through A-type K channels by subtraction. Isoflurane at clinically relevant concentrations accelerated the decay of the A-type K current and delayed the recovery from inactivation without changing the steady-state inactivation curves. Isoflurane did not affect the non-inactivating outward current. Addition of 4-aminopyridine partially occluded the excitatory effects of isoflurane in current-clamp recordings. These results demonstrate that isoflurane accelerated the inactivation and delayed the recovery from inactivation of A-type K channels in principal neurons in rat substantia nigra pars compacta without affecting delayed rectifier K channels. These effects may contribute in part to excitation of these neurons and the isoflurane-induced increases in dopamine release reported in vitro and in vivo.

Microglia are the main immunocompetent cells of the mammalian central nervous system (CNS). Activation of cultured microglial cells and subsequent release of nitric oxide and cytokines critically depends on intracellular calcium levels. Since microglia undergo dramatic morphological, biochemical and electrophysiological changes in response to pathological events in the CNS, we investigated temporal changes in expression levels of ion channels involved in cellular calcium homeostasis in mouse cortical microglial cells in culture. Specifically, we assessed the inward and delayed outward rectifier potassium currents (I IRK and I DRK), calcium (Ca2+) release-activated Ca2+ currents (I CRAC) and Ca2+-activated TRPM4-like currents (I CAN) in non-activated microglia and cells that were activated by exposure to lipopolysaccharide (LPS) between 3 and 48 h. Unstimulated microglial cells, subcultured from an astrocyte coculture, typically exhibited a ramified, rod-shaped morphology. During the first 3 days of culture cell size and shape were maintained, but the percentage of cells showing prominent I IRK went up and those expressing I DRK went down. Cells retaining I DRK exhibited smaller amplitudes, whereas those of I IRK and I CRAC were not affected. However, after 24 h of exposure to 1 microg ml(-1) LPS, most cells showed an amoeboid ('fried egg'-shaped) morphology with a 62% increase in cell capacitance. At that point in time, only 14% of the cells revealed I IRK and 3% had I DRK exclusively, whereas the majority of cells expressed both currents. The amplitudes of I CRAC and I IRK progressively decreased after stimulation, whereas I DRK transiently reached a maximum after 6 h of LPS exposure and then returned to pre-stimulation expression levels. Cultured microglia also revealed TRPM4-like, Ca2+-activated non-selective currents (I CAN) with an EC50 of 1.2 microm [Ca2+]i. The expression levels of this current did not change significantly during and after 24 h of LPS exposure. We propose that LPS-induced down-regulation of I IRK and I CRAC will reduce the cell's capacity to produce significant calcium influx upon receptor activation and result in decreased sensitivity to exogenous stimulation. In this scenario, I CAN expression would remain constant, although its activity would automatically be reduced due to the diminished calcium influx capacity of the cell.

Background: QT prolongation is an incomplete measure of drug-induced changes in repolarization. In this study, we investigated a novel, automatic ECG technique for describing ventricular repolarization morphology and we compared these results to corrected QT (QTc) prolongation for identifying ECGs of healthy individuals on moxifloxacin., Methods: We analysed data from the US FDA ECG Warehouse involving 160 standard ECGs from 40 healthy subjects enrolled in a randomized, parallel, placebo-controlled, 'thorough QT' study. Computerized ECG analysis included a series of scalar and vectorial parameters describing duration of repolarization segments and T-wave/loop morphology including its symmetry, amplitude and shape. Binary logistic models for the identification of moxifloxacin-induced abnormalities of the repolarization were developed., Results: Moxifloxacin induced significant changes in several ECG parameters including QT and QT apex and early repolarization duration (ERD)(30)(%), T-wave amplitude and slopes of the ascending and descending arm of the T-wave. The logistic model based only on T-wave morphology parameters outperformed the model based on QTc interval for identifying the presence of moxifloxacin. Combining information about repolarization interval duration with T-wave morphology significantly improved the detection of presence of moxifloxacin (p < 0.01). The increased sensitivity of our novel ECG method contributes to a >40% reduction in the sample size required to detect significant QTc prolongation induced by moxifloxacin., Conclusions: Repolarization morphology is significantly altered by moxifloxacin. The computerized ECG technique provides a novel method for quantifying morphological changes of repolarization segment. Our new parameters reflecting the morphology of the T-wave outperformed QTc measurements when identifying moxifloxacin-induced blockade of the outward rapid components of the delayed rectifier repolarizing potassium current (I(Kr)). These data indicate that the analysis of T-wave morphology could play a role in the assessment of drug toxicity.

Background and Purpose: I(Kur) (Ultra-rapid delayed rectifier current) has microM sensitivity to 4-aminopyridine (4-AP) and is an important modulator of the plateau amplitude and action potential duration in canine atria. Kv1.5 encodes I(Kur) and is present in both atria and ventricles in canines and humans. We hypothesized that a similar plateau outward current with microM sensitivity to 4-AP is present in canine ventricle., Experimental Approach: We used established voltage clamp protocols and used 4-AP (50 and 100 microM) to measure a plateau outward current in normal canine myocytes isolated from the left ventricular mid-myocardium., Key Results: Action potential recordings in the presence of 4-AP showed significant prolongation of action potential duration at 50 and 90% repolarization at 0.5 and 1 Hz (P<0.05), while no prolongation occurred at 2 Hz. Voltage clamp experiments revealed a rapidly activating current, similar to current characteristics of canine atrial I(Kur), in approximately 70% of left ventricular myocytes. The IC(50) of 4-AP for this current was 24.2 microM. The concentration of 4-AP used in our experiments resulted in selective blockade of an outward current that was not I(to) or I(Kr). Beta-adrenergic stimulation with isoprenaline significantly increased the 4-AP sensitive outward current density (P<0.05), suggesting a role for this current during increased sympathetic stimulation. In silico incorporation into a canine ventricular cell model revealed selective AP prolongation after current blockade., Conclusions and Implications: Our results support the existence of a canine ventricular plateau outward current sensitive to micromolar 4-AP and its constitutive role in ventricular repolarization.

Background and Purpose: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases., Experimental Approach: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities., Key Results: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current., Conclusions and Implications: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.

Prolongation of the QT interval has been observed during treatment with olanzapine, a thienobenzodiazepine antipsychotic agent. Our objectives were 1) to characterize the effects of olanzapine on cardiac repolarization and 2) to evaluate effects of olanzapine on the major time-dependent outward potassium current involved in cardiac repolarization, namely I(Kr) (I(Kr): rapid component of the delayed rectifier potassium current).Isolated, buffer-perfused guinea pig hearts (n = 40) were stimulated at different pacing cycle lengths (150-250 msec) and exposed to olanzapine at concentrations ranging from 1 to 100 microM. Olanzapine increased monophasic action potential duration measured at 90% repolarization (MAPD90) in a concentration-dependent manner by 6.7 +/- 0.7 msec at 3 microM but by 26.0 +/- 4.3 msec at 100 microM (250 msec cycle length). Increase in MAPD(90) was also reverse frequency dependent; 30 microM olanzapine increased MAPD90 by 28.0 +/- 6.2 msec at a pacing cycle length of 250 msec but by only 18.9 +/- 2.2 msec at a pacing cycle length of 150 msec. Experiments in HERG-transfected (HERG: human ether-a-gogo-related gene) HEK293 cells (n = 36) demonstrated concentration-dependent block of the rapid component (I(Kr)) of the delayed rectifier potassium current: tail current was decreased 50% at olanzapine 3.8 microM. Olanzapine possesses direct cardiac electrophysiological effects similar to those of class III anti-arrhythmic drugs. These effects were observed at concentrations that can be measured in patients under conditions of impaired drug elimination such as renal or hepatic insufficiency, during co-administration of other CYP1A2 substrates/inhibitors or after drug overdose. These results offer a new potential explanation for QT prolonging effects observed during olanzapine treatment in patients.