Evidence for Inhibitory Perturbations on the Amplitude, Gating, and Hysteresis of A-Type Potassium Current, Produced by Lacosamide, a Functionalized Amino Acid with Anticonvulsant Properties.

Lacosamide (Vimpat®, LCS) is widely known as a functionalized amino acid with promising anti-convulsant properties; however, adverse events during its use have gradually appeared. Despite its inhibitory effect on voltage-gated Na+ current (INa), the modifications on varying types of ionic currents caused by this drug remain largely unexplored. In pituitary tumor (GH3) cells, we found that the presence of LCS concentration-dependently decreased the amplitude of A-type K+ current (IK(A)) elicited in response to membrane depolarization. The IK(A) amplitude in these cells was sensitive to attenuation by the application of 4-aminopyridine, 4-aminopyridine-3-methanol, or capsaicin but not by that of tetraethylammonium chloride. The effective IC50 value required for its reduction in peak or sustained IK(A) was calculated to be 102 or 42 µM, respectively, while the value of the dissociation constant (KD) estimated from the slow component in IK(A) inactivation at varying LCS concentrations was 52 µM. By use of two-step voltage protocol, the presence of this drug resulted in a rightward shift in the steady-state inactivation curve of IK(A) as well as in a slowing in the recovery time course of the current block; however, no change in the gating charge of the inactivation curve was detected in its presence. Moreover, the LCS addition led to an attenuation in the degree of voltage-dependent hysteresis for IK(A) elicitation by long-duration triangular ramp voltage commands. Likewise, the IK(A) identified in mouse mHippoE-14 neurons was also sensitive to block by LCS, coincident with an elevation in the current inactivation rate. Collectively, apart from its canonical action on INa inhibition, LCS was effective at altering the amplitude, gating, and hysteresis of IK(A) in excitable cells. The modulatory actions on IK(A), caused by LCS, could interfere with the functional activities of electrically excitable cells (e.g., pituitary tumor cells or hippocampal neurons).

Suppression of delayed rectifier K+ channels by gentamicin induces membrane hyperexcitability through JNK and PKA signaling pathways in vestibular ganglion neurons.

Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.

Characterization of the Synergistic Inhibition of IK(erg) and IK(DR) by Ribociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor.

Ribociclib (RIB, LE011, Kisqali®), an orally administered inhibitor of cyclin-dependent kinase-4/6 (CDK-4/6) complex, is clinically effective for the treatment of several malignancies, including advanced breast cancer. However, information regarding the effects of RIB on membrane ion currents is limited. In this study, the addition of RIB to pituitary tumor (GH3) cells decreased the peak amplitude of erg-mediated K+ current (IK(erg)), which was accompanied by a slowed deactivation rate of the current. The IC50 value for RIB-perturbed inhibition of deactivating IK(erg) in these cells was 2.7 µM. In continued presence of µM RIB, neither the subsequent addition of 17ß-estradiol (30 µM), phorbol 12-myristate 13-acetate (10 µM), or transforming growth factor-ß (1 µM) counteracted the inhibition of deactivating IK(erg). Its presence affected the decrease in the degree of voltage-dependent hysteresis for IK(erg) elicitation by long-duration triangular ramp voltage commands. The presence of RIB differentially inhibited the peak or sustained component of delayed rectifier K+ current (IK(DR)) with an effective IC50 of 28.7 or 11.4 µM, respectively, while it concentration-dependently decreased the amplitude of M-type K+ current with IC50 of 13.3 µM. Upon 10-s long membrane depolarization, RIB elicited a decrease in the IK(DR) amplitude, which was concomitant with an accelerated inactivation time course. However, the inability of RIB (10 µM) to modify the magnitude of the hyperpolarization-activated cation current was disclosed. The mean current-voltage relationship of IK(erg) present in HL-1 atrial cardiomyocytes was inhibited in the presence of RIB (10 µM). Collectively, the hyperpolarization-activated cation current was observed. RIB-mediated perturbations in ionic currents presented herein are upstream of its suppressive action on cytosolic CDK-4/6 activities and partly participates in its modulatory effects on the functional activities of pituitary tumor cells (e.g., GH3 cells) or cardiac myocytes (e.g., HL-1 cells).

Effectiveness of nalbuphine, a κ-opioid receptor agonist and µ-opioid receptor antagonist, in the inhibition of INa , IK(M) , and IK(erg) unlinked to interaction with opioid receptors.

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the µ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na+ current (INa ) with an IC50 value of 1.9 µM. It shifted the steady-state inactivation curve of peak INa to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak INa . In continued presence of NAL, subsequent application of either dynorphin A1-13 (1 µM) or naloxone (30 µM) failed to modify its suppression of peak INa . Tefluthrin (Tef; 10 µM), a pyrethroid known to activate INa , increased peak INa with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak INa followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K+ current [IK(M) ] with an IC50 value of 5.7 µM, while it slightly suppressed erg-mediated and delayed-rectifier K+ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca2+ -activated K+ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak INa , and subsequent addition of Tef reversed NAL-induced suppression of INa . Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including INa and IK(M) , thereby influencing the functional activities of central neurons.

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells.

BACKGROUND: Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. METHODS: Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH3) cells were evaluated by patch clamp techniques. RESULTS: ART inhibited the amplitude of delayed-rectifier K+ current (IK(DR)) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on IK(DR) with an IC50 value of 11.2 µM and enhanced IK(DR) inactivation with a KD value of 14.7 µM. The steady-state inactivation curve of IK(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of IK(DR) in GH3 cells. ART also decreased the peak amplitude of voltage-gated Na+ current (INa) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late INa, was effective at reversing ART-induced prolongation in inactivation time constant of INa. Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. CONCLUSION: Under ART exposure, the inhibitory actions on both IK(DR) and INa could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

BACKGROUND AND PURPOSE: The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACH: Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTS: Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30 µM genistein or 10 µM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 µM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONS: The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .

Molecular basis and drug sensitivity of the delayed rectifier (IKr) in the fish heart.

Fishes are increasingly used as models for human cardiac diseases, creating a need for a better understanding of the molecular basis of fish cardiac ion currents. To this end we cloned KCNH6 channel of the crucian carp (Carassius carassius) that produces the rapid component of the delayed rectifier K(+) current (IKr), the main repolarising current of the fish heart. KCNH6 (ccErg2) was the main isoform of the Kv11 potassium channel family with relative transcript levels of 98.9% and 99.6% in crucian carp atrium and ventricle, respectively. KCNH2 (ccErg1), an orthologue to human cardiac Erg (Herg) channel, was only slightly expressed in the crucian carp heart. The native atrial IKr and the cloned ccErg2 were inhibited by similar concentrations of verapamil, terfenadine and KB-R7943 (P>0.05), while the atrial IKr was about an order of magnitude more sensitive to E-4031 than ccErg2 (P<0.05) suggesting that some accessory ß-subunits may be involved. Sensitivity of the crucian carp atrial IKr to E-4031, terfenadine and KB-R7943 was similar to what has been reported for the Herg channel. In contrast, the sensitivity of the crucian carp IKr to verapamil was approximately 30 times higher than the previously reported values for the Herg current. In conclusion, the cardiac IKr is produced by non-orthologous gene products in fish (Erg2) and mammalian hearts (Erg1) and some marked differences exist in drug sensitivity between fish and mammalian Erg1/2 which need to be taken into account when using fish heart as a model for human heart.

ß1-adrenergic regulation of rapid component of delayed rectifier K+ currents in guinea-pig cardiac myocytes.

Human ether-à-go-go-related gene (hERG) potassium channels conduct the rapid component of the delayed rectifier potassium current (IKr), which is crucial for repolarization of cardiac action potential. Patients with hERG­associated long QT syndrome usually develop tachyarrhythmias during physical and/or emotional stress, both known to stimulate adrenergic receptors. The present study aimed to investigate a putative functional link between ß1-adrenergic stimulation and IKr in guinea-pig left ventricular myocytes and to analyze how IKr is regulated following activation of the ß1-adrenergic signaling pathway. The IKr current was measured using a whole-cell patch-clamp technique. A selective ß1-adrenergic receptor agonist, xamoterol, at concentrations of 0.01-100 µM decreased IKr in a concentration-dependent manner. The 10 µM xamoterol-induced inhibition of IKr was attenuated by the protein kinase A (PKA) inhibitor KT5720, the protein kinase C (PKC) inhibitor chelerythrine, and the phospholipase (PLC) inhibitor U73122, indicating involvement of PKA, PKC and PLC in ß1-adrenergic inhibition of IKr. The results of the present study indicate an association between IKr and the ß1-adrenergic receptor in arrhythmogenesis, involving the activation of PKA, PKC and PLC.

Crude oil impairs cardiac excitation-contraction coupling in fish.

Crude oil is known to disrupt cardiac function in fish embryos. Large oil spills, such as the Deepwater Horizon (DWH) disaster that occurred in 2010 in the Gulf of Mexico, could severely affect fish at impacted spawning sites. The physiological mechanisms underlying such potential cardiotoxic effects remain unclear. Here, we show that crude oil samples collected from the DWH spill prolonged the action potential of isolated cardiomyocytes from juvenile bluefin and yellowfin tunas, through the blocking of the delayed rectifier potassium current (I(Kr)). Crude oil exposure also decreased calcium current (I(Ca)) and calcium cycling, which disrupted excitation-contraction coupling in cardiomyocytes. Our findings demonstrate a cardiotoxic mechanism by which crude oil affects the regulation of cellular excitability, with implications for life-threatening arrhythmias in vertebrates.

The actions of mdivi-1, an inhibitor of mitochondrial fission, on rapidly activating delayed-rectifier K⁺ current and membrane potential in HL-1 murine atrial cardiomyocytes.

Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (I(tail)) for the rapidly activating delayed-rectifier K⁺ current (I(Kr)) in a concentration-dependent manner with an IC50 value at 11.6 µM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of I(tail) to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K⁺ channels without modifying the single-channel conductance. Mdivi-1 (30 µM) slightly depressed the peak amplitude of Na⁺ current with no change in the overall current-voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH3 cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K⁺ current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K⁺ channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division.

Evidence that endogenous hydrogen sulfide exerts an excitatory effect on gastric motility in mice.

The present study was designed to investigate the effect of endogenous hydrogen sulfide (H2S) on gastric motility in mice. Western blotting and immunocytochemistry were used to determine expression levels of the H2S-producing enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) in gastric tissues and cultured smooth muscle cells. Physiological and intracellular recordings and the whole-cell patch clamp technique were used to evaluate the effect of H2S on the mechanical and electrical activities in muscle strips and in isolated smooth muscle cells, respectively. The results showed that CBS and CSE were expressed in mouse gastric smooth muscle. NaHS, a H2S donor, inhibited the amplitude and frequency of spontaneous contraction at high concentrations (>200 µM). However, NaHS at low concentrations (<100 µM) enhanced the basal tension and increased the contractile amplitude of muscle strips. This excitatory effect was not altered by the blockade of the enteric nerve with TTX, but was abolished by tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Aminooxyacetic acid (AOA), but not propargylglycine (PAG), caused a concentration-dependent inhibition of spontaneous contraction. This effect was restored by L-cysteine and NaHS. In addition, NaHS at low concentrations (<100 µM) produced a depolarization of the membrane potential, whereas AOA hyperpolarized the membrane potential and decreased the amplitude of slow waves. Furthermore, AOA increased whole-cell delayed rectifier K⁺ current (I(K(V))). These findings suggest that endogenous H2S appears to be an excitatory gaseous mediator during physiological regulation of gastric motility and this excitable effect is mediated by depolarization of the membrane potential via inhibition of I(K(V)).

Evidence for aconitine-induced inhibition of delayed rectifier K(+) current in Jurkat T-lymphocytes.

Aconitine (ACO) is a highly toxic diterpenoid alkaloid and known to exert the immunomodulatory action. However, whether it has any effects on ion currents in immune cells remains unknown. The effects of ACO and other related compounds on ion currents in Jurkat T-lymphocytes were investigated in this study. ACO suppressed the amplitude of delayed-rectifier K(+) current (I(K(DR))) in a time- and concentration-dependent manner. Margatoxin (100 nM), a specific blocker of K(V)1.3-encoded current, decreased the I(K(DR)) amplitude in these cells and the ACO-induced inhibition of I(K(DR)) was not reversed by 1-ethyl-2-benzimidazolinone (30 µM) or nicotine (10 µM). The IC(50) value for ACO-mediated inhibition of I(K(DR)) was 5.6 µM. ACO accelerated the inactivation of I(K(DR)) with no change in the activation rate of this current. Increasing the ACO concentration not only reduced the I(K(DR)) amplitude, but also accelerated the inactivation time course of the current. With the aid of minimal binding scheme, the inhibitory action of ACO on I(K(DR)) was estimated with a dissociation constant of 6.8 µM. ACO also shifted 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 enhanced in the presence of ACO. In Jurkat cells incubated with amiloride (30 µM), the ACO-induced inhibition of I(K(DR)) remained unaltered. In RAW 264.7 murine macrophages, ACO did not modify the kinetics of I(K(DR)), although it suppressed I(K(DR)) amplitude. Taken together, these effects can significantly contribute to its action on functional activity of immune cells if similar results are found in vivo.

Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents.

Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 µM) suppressed I(Kr) in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 µM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 µM bisindolylmaleimide I or 0.1 µM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution of PKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.

The selective estrogen receptor modulator raloxifene inhibits cardiac delayed rectifier potassium currents and voltage-gated sodium current without QTc interval prolongation.

Raloxifene is widely used in the treatment of postmenopausal osteoporosis and also has been shown to be cardioprotective. The effect of raloxifene on cardiac ion channels is not fully understood. The present study investigated whether raloxifene could affect the cloned hERG channel (I(hERG)) and recombinant human cardiac KCNQ1/KCNE1 channel (I(Ks)) stably expressed in HEK 293 cells using a patch-clamp technique. Raloxifene blocked I(hERG) with an IC(50) of 1.1 µM and decreased I(Ks) (IC(50): 4.8 µM) without affecting activation kinetics. In addition, raloxifene significantly decreased I(Na) (IC(50): 2.8 µM) in guinea pig ventricular myocytes. However, this drug (1 µM) did not increase QRS and QTc interval in isolated guinea pig hearts. These results demonstrate that raloxifene, despite its inhibitory action on delayed rectifier potassium currents, does not prolong ECG QTc interval, suggesting that raloxifene is likely a safe selective estrogen receptor modulator with less cardiac toxicity.

Electrophysiological properties of human induced pluripotent stem cells.

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC(50) = 3.3 +/- 2.7 mM) delayed rectifier K(+) currents (I(KDR)) in 105 of 110 single iPSCs (15.4 +/- 0.9 pF). I(KDR) in iPSCs displayed a current density of 7.6 +/- 3.8 pA/pF at +40 mV. The voltage for 50% activation (V(1/2)) was -7.9 +/- 2.0 mV, slope factor k = 9.1 +/- 1.5. However, Ca(2+)-activated K(+) current (I(KCa)), hyperpolarization-activated pacemaker current (I(f)), and voltage-gated sodium channel (Na(V)) and voltage-gated calcium channel (Ca(V)) currents could not be measured. TEA inhibited iPSC proliferation (EC(50) = 7.8 +/- 1.2 mM) and viability (EC(50) = 5.5 +/- 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC(50) = 4.5 +/- 0.5 mM) but had less effect on proliferation (EC(50) = 0.9 +/- 0.5 mM). Cell cycle analysis further revealed that K(+) channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of I(KCa) in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.

Paroxysmal beta-adrenergic receptor-mediated alterations in ventricular repolarization at rapid heart rates during inhibition of delayed rectifier currents.

The contribution of the slow component of the delayed rectifier current (IKs) to ventricular repolarization is increased during rapid heart rates and prolonged repolarization. The objective was to characterize physiologically relevant paroxysmal beta-adrenergic receptor-mediated alterations on ventricular repolarization under these conditions. Paced guinea pig hearts were perfused with (1) control, (2) sparfloxacin (IKr inhibitor), or (3) sparfloxacin and HMR 1556 (IKs inhibitor). The mean +/- standard error of the mean epicardial action potential duration at 90% repolarization (APD90) increased from baseline with IKr inhibition (12.9% +/- 4.7%) and dual IKr/IKs inhibition (25.1% +/- 5.3). Paroxysmal isoproterenol (0.01 and 1.0 nM) significantly decreased APD90 in the presence of IKr inhibition but was attenuated with the addition of IKs inhibition. Spontaneous episodes of polymorphic ventricular tachycardia were observed with isoproterenol during dual IKr and IKs inhibition. The endocardial expression of KCNQ1 increased greater than 2-fold after exposure to IKr and dual IKr/IKs inhibition relative to control but was not altered in epicardial tissue. The beta-adrenergic receptor-mediated decrease in APD90 during IKr inhibition is reversed in the presence of IKs inhibition at rapid heart rates. IKs may serve as an important compensatory mechanism to protect against adrenergically induced arrhythmias when the repolarization reserve is depleted.

Methadone-induced mortality in the treatment of chronic pain: role of QT prolongation.

Methadone is increasingly prescribed for chronic pain, yet the associated mortality appears to be rising disproportionately relative to other opioid analgesics. We review the available evidence on methadone-associated mortality, and explore potential pharmacokinetic and pharmacodynamic explanations for its greater apparent lethality. While methadone shares properties of central nervous system and respiratory depression with other opioids, methadone is unique as a potent blocker of the delayed rectifier potassium ion channel (IKr). This results in QT-prolongation and torsade de pointes (TdP) in susceptible individuals. In some individuals with low serum protein binding of methadone, the extent of blockade is roughly comparable to that of sotalol, a potent QT-prolonging drug. Predicting an individual's propensity for methadone-induced TdP is difficult at present given the inherent limitations of the QT interval as a risk-stratifier combined with the multifactorial nature of the arrhythmia. Consensus recommendations have recently been published to mitigate the risk of TdP until further studies better define the arrhythmia risk factors for methadone. Studies are needed to provide insights into the clinical covariates most likely to result in methadone-associated arrhythmia and to assess the feasibility of current risk mitigation strategies.

Effects of methylphenidate hydrochloride on the cardiovascular system in vivo and in vitro: a safety pharmacology study.

INTRODUCTION: : We examined the effects of methylphenidate hydrochloride (MPH) on the cardiovascular system using in vivo and in vitro study methods in accordance with the ICH-S7B guideline. METHODS: MPH was orally administered at doses of 3, 10 and 30 mg/kg to unrestrained conscious dogs implanted with a telemetry transmitter and attached with body surface electrodes, and electrocardiogram (ECG) leads. The QTcF interval was determined while heart rate (HR), and blood pressure (BP) were measured. Action potentials in isolated guinea-pig papillary muscle and the rapid component of the delayed rectifier potassium current (I(Kr)) in HEK-293 cells stably transfected with hERG were also investigated at concentrations of 0.1, 0.3 and 1 microg/mL (0.37, 1.1 and 3.7 micromol/L) of MPH. RESULTS: No ECG changes were observed except for a shortening of the QT interval due to a shortening of the RR interval at the maximum dose tested, 30 mg/kg. The only observed change was an elevation of BP in dogs at the dose of 30 mg/kg, which is approximately 10 times higher than the maximum therapeutic dose for use in children with attention deficit hyperactivity disorder (ADHD). Neither APD prolongation nor I(Kr) inhibition was observed by MPH in the in vitro studies up to the maximum concentration tested, 1 microg/mL (3.7 micromol/L), which is approximately 34 times higher than the clinically attainable unbound plasma MPH concentrations in children with ADHD. DISCUSSION: These results suggest that it is unlikely that MPH affects ventricular repolarization processes at the therapeutically recommended dose levels in patients with ADHD.

Experimental and simulation studies on the mechanisms of levetiracetam-mediated inhibition of delayed-rectifier potassium current (KV3.1): contribution to the firing of action potentials.

Levetiracetam (LEV) is an S-enantiomer pyrrolidone derivative with established antiepileptic efficacy in generalized epilepsy and partial epilepsy. However, its effects on ion currents and membrane potential remain largely unclear. We investigated the effect of LEV on differentiated NG108-15 neurons. In these cells treated with dibutyryl cyclic AMP, the expression level of the K(V)3.1 mRNA was elevated. With the aid of patch clamp technology, we found that LEV could suppress the amplitude of delayed rectifier K(+) current (I(K(DR))) in a concentration-dependent manner with an IC(50) value of 37 microM. LEV (30 microM) shifted the steady-state activation of I(K(DR)) to a more positive potential by 10 mV, without shifting the steady-state inactivation of I(K(DR)). Neither Na(+), nor erg (ether-a-go-go-related)-mediated K(+) and ATP-sensitive K(+) currents were affected by LEV (100 microM). LEV increased the duration of action potentials in current clamp configuration. Simulation studies in a modified Hodgkin-Huxley neuron and network unraveled that the reduction of slowly inactivating I(K(DR)) resulted in membrane depolarization accompanied by termination of the firing of action potentials in a stochastic manner. Therefore, the inhibitory effects on slowly inactivating I(K(DR)) (K(V)3.1-encoded current) may constitute one of the underlying mechanisms through which LEV affect neuronal activity in vivo.

The hERG potassium channel and hERG screening for drug-induced torsades de pointes.

Drug-induced torsades de pointes (TdP) arrhythmia is a major safety concern in the process of drug design and development. The incidence of TdP tends to be low, so early pre-clinical screens rely on surrogate markers of TdP to highlight potential problems with new drugs. hERG (human ether-à-go-go-related gene, alternative nomenclature KCNH2) is responsible for channels mediating the 'rapid' delayed rectifier K+ current (IKr) which plays an important role in ventricular repolarization. Pharmacological inhibition of native IKr and of recombinant hERG channels is a shared feature of diverse drugs associated with TdP. In vitro hERG assays therefore form a key element of an integrated assessment of TdP liability, with patch-clamp electrophysiology offering a 'gold standard'. However, whilst clearly necessary, hERG assays cannot be assumed automatically to provide sufficient information, when considered in isolation, to differentiate 'safe' from 'dangerous' drugs. Other relevant factors include therapeutic plasma concentration, drug metabolism and active metabolites, severity of target condition and drug effects on other cardiac ion channels that may mitigate or exacerbate effects of hERG blockade. Increased understanding of the nature of drug-hERG channel interactions may ultimately help eliminate potential hERG blockade early in the design and development process. Currently, for promising drug candidates integration of data from hERG assays with information from other pre-clinical safety screens remains essential.