Midazolam's Effects on Delayed-Rectifier K+ Current and Intermediate-Conductance Ca2+-Activated K+ Channel in Jurkat T-lymphocytes.

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 µM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 µM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.

Inhibitory Effective Perturbations of Cilobradine (DK-AH269), A Blocker of HCN Channels, on the Amplitude and Gating of Both Hyperpolarization-Activated Cation and Delayed-Rectifier Potassium Currents.

Cilobradine (CIL, DK-AH269), an inhibitor of hyperpolarization-activated cation current (Ih), has been observed to possess pro-arrhythmic properties. Whether and how CIL is capable of perturbing different types of membrane ionic currents existing in electrically excitable cells, however, is incompletely understood. In this study, we intended to examine possible modifications by it or other structurally similar compounds of ionic currents in pituitary tumor (GH3) cells and in heart-derived H9c2 cells. The standard whole-cell voltage-clamp technique was performed to examine the effect of CIL on ionic currents. GH3-cell exposure to CIL suppressed the density of hyperpolarization-evoked Ih in a concentration-dependent manner with an effective IC50 of 3.38 µM. Apart from its increase in the activation time constant of Ih during long-lasting hyperpolarization, the presence of CIL (3 µM) distinctly shifted the steady-state activation curve of Ih triggered by a 2-s conditioning pulse to a hyperpolarizing direction by 10 mV. As the impedance-frequency relation of Ih was studied, its presence raised the impedance magnitude at the resonance frequency induced by chirp voltage. CIL also suppressed delayed-rectifier K+ current (IK(DR)) followed by the accelerated inactivation time course of this current, with effective IC50 (measured at late IK(DR)) or KD value of 3.54 or 3.77 µM, respectively. As the CIL concentration increased 1 to 3 µM, the inactivation curve of IK(DR) elicited by 1- or 10-s conditioning pulses was shifted to a hyperpolarizing potential by approximately 10 mV, and the recovery of IK(DR) inactivation during its presence was prolonged. The peak Na+ current (INa) during brief depolarization was resistant to being sensitive to the presence of CIL, yet to be either decreased by subsequent addition of A-803467 or enhanced by that of tefluthrin. In cardiac H9c2 cells, unlike the CIL effect, the addition of either ivabradine or zatebradine mildly led to a lowering in IK(DR) amplitude with no conceivable change in the inactivation time course of the current. Taken together, the compound like CIL, which was tailored to block hyperpolarization-activated cation (HCN) channels effectively, was also capable of altering the amplitude and gating of IK(DR), thereby influencing the functional activities of electrically excitable cells, such as GH3 cells.

High Capability of Pentagalloylglucose (PGG) in Inhibiting Multiple Types of Membrane Ionic Currents.

Pentagalloyglucose (PGG, penta-O-galloyl-ß-d-glucose; 1,2,3,4,6-pentagalloyl glucose), a pentagallic acid ester of glucose, is recognized to possess anti-bacterial, anti-oxidative and anti-neoplastic activities. However, to what extent PGG or other polyphenolic compounds can perturb the magnitude and/or gating of different types of plasmalemmal ionic currents remains largely uncertain. In pituitary tumor (GH3) cells, we found out that PGG was effective at suppressing the density of delayed-rectifier K+ current (IK(DR)) concentration-dependently. The addition of PGG could suppress the density of proton-activated Cl- current (IPAC) observed in GH3 cells. The IC50 value required for the inhibitory action of PGG on IK(DR) or IPAC observed in GH3 cells was estimated to be 3.6 or 12.2 µM, respectively, while PGG (10 µM) mildly inhibited the density of the erg-mediated K+ current or voltage-gated Na+ current. The presence of neither chlorotoxin, hesperetin, kaempferol, morin nor iberiotoxin had any effects on IPAC density, whereas hydroxychloroquine or 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5yl)oxy] butanoic acid suppressed current density effectively. The application of PGG also led to a decrease in the area of voltage-dependent hysteresis of IPAC elicited by long-lasting isosceles-triangular ramp voltage command, suggesting that hysteretic strength was lessened in its presence. In human cardiac myocytes, the exposure to PGG also resulted in a reduction of ramp-induced IK(DR) density. Taken literally, PGG-perturbed adjustment of ionic currents could be direct and appears to be independent of its anti-oxidative property.

Actions of FTY720 (Fingolimod), a Sphingosine-1-Phosphate Receptor Modulator, on Delayed-Rectifier K+ Current and Intermediate-Conductance Ca2+-Activated K+ Channel in Jurkat T-Lymphocytes.

FTY720 (fingolimod), a modulator of sphingosine-1-phosphate receptors, is known to produce the immunomodulatory actions and to be beneficial for treating the relapsing multiple sclerosis. However, whether it exerts any effects on membrane ion currents in immune cells remains largely unknown. Herein, the effects of FTY720 on ionic currents in Jurkat T-lymphocytes were investigated. Cell exposure to FTY720 suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner with an IC50 value of 1.51 µM. Increasing the FTY720 concentration not only decreased the IK(DR) amplitude but also accelerated the inactivation time course of the current. By using the minimal reaction scheme, the effect of FTY720 on IK(DR) inactivation was estimated with a dissociation constant of 3.14 µM. FTY720 also shifted the inactivation curve of IK(DR) to a hyperpolarized potential with no change in the slope factor, and recovery from IK(DR) became slow during the exposure to this compound. Cumulative inactivation for IK(DR) in response to repetitive depolarizations was enhanced in the presence of FTY720. In SEW2871-treated cells, FTY720-induced inhibition of IK(DR) was attenuated. This compound also exerted a stimulatory action on the activity of intermediate-conductance Ca2+-activated K+ channels in Jurkat T-lymphocytes. However, in NSC-34 neuronal cells, FTY720 did not modify the inactivation kinetics of KV3.1-encoded IK(DR), although it suppressed IK(DR) amplitude in these cells. Collectively, the perturbations by FTY720 on different types of K+ channels may contribute to the functional activities of immune cells, if similar findings appear in vivo.

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.

Evidence for the Capability of Roxadustat (FG-4592), an Oral HIF Prolyl-Hydroxylase Inhibitor, to Perturb Membrane Ionic Currents: An Unidentified yet Important Action.

Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 µM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 µM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.

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.

Important modifications by sugammadex, a modified γ-cyclodextrin, of ion currents in differentiated NSC-34 neuronal cells.

BACKGROUND: Sugammadex (SGX) is a modified γ-cyclodextrin used for reversal of steroidal neuromuscular blocking agents during general anesthesia. Despite its application in clinical use, whether SGX treatment exerts any effects on membrane ion currents in neurons remains largely unclear. In this study, effects of SGX treatment on ion currents, particularly on delayed-rectifier K+ current [I K(DR)], were extensively investigated in differentiated NSC-34 neuronal cells. RESULTS: After cells were exposed to SGX (30 µM), there was a reduction in the amplitude of I K(DR) followed by an apparent slowing in current activation in response to membrane depolarization. The challenge of cells with SGX produced a depolarized shift by 15 mV in the activation curve of I K(DR) accompanied by increased gating charge of this current. However, the inactivation curve of I K(DR) remained unchanged following SGX treatment, as compared with that in untreated cells. According to a minimal reaction scheme, the lengthening of activation time constant of I K(DR) caused by cell treatment with different SGX concentrations was quantitatively estimated with a dissociation constant of 17.5 µM, a value that is clinically achievable. Accumulative slowing in I K(DR) activation elicited by repetitive stimuli was enhanced in SGX-treated cells. SGX treatment did not alter the amplitude of voltage-gated Na+ currents. In SGX-treated cells, dexamethasone (30 µM), a synthetic glucocorticoid, produced little or no effect on L-type Ca2+ currents, although it effectively suppressed the amplitude of this current in untreated cells. CONCLUSIONS: The treatment of SGX may influence the amplitude and gating of I K(DR) and its actions could potentially contribute to functional activities of motor neurons if similar results were found in vivo.

APOEε4 increases trauma induced early apoptosis via reducing delayed rectifier K(+) currents in neuronal/glial co-cultures model.

Traumatic brain injury (TBI) is a commonly encountered emergency and severe neurosurgical injury. Previous studies have shown that the presence of the apolipoprotein E (APOE) ε4 allele has adverse outcomes across the spectrum of TBI severity. Our objective was to evaluate the effects of APOE alleles on trauma induced early apoptosis via modification of delayed rectifier K(+) current (Ik(DR)) in neuronal/glial co-cultures model. An ex vivo neuronal/glial co-cultures model carrying individual APOE alleles (ε2, ε3, ε4) of mechanical injury was developed. Flow cytometry and patch clamp recording were performed to analyze the correlations among APOE genotypes, early apoptosis and Ik(DR). We found that APOEε4 increased early apoptosis at 24h (p<0.05) compared to the ones transfected with APOEε3 and APOEε2. Noticeably, APOEε4 significantly reduced the amplitude of the Ik(DR) at 24h compared to the APOEε3 and APOEε2 (p<0.05) which exacerbate Ca(2+) influx. This indicates a possible effect of APOEε4 on early apoptosis via inhibiting Ik(DR) following injury which may adversely affect the outcome of TBI.

Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes.

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K(+) channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K(+) current (Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K(+) current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.

An unidentified yet notable modification on I Na and I K (DR) caused by ramelteon.

Despite advancement in anti-seizure medications, 30% of patients continue to experience recurrent seizures. Previous data indicated the antiepileptic properties of melatonin and its agonists in several animal models. However, the underlying mechanisms of melatonin and its agonists on cellular excitability remain poorly understood. In this study, we demonstrated the electrophysiological changes of two main kinds of ion channels that are responsible for hyperexcitability of neurons after introduction of melatonin agonists- ramelteon (RAM). In Neuro-2a cells, the amplitude of voltage-gated Na+ (I Na) and delayed-rectifier K+ currents (I K (DR)) could be suppressed under RAM. The IC50 values of 8.7 and 2.9 µM, respectively. RAM also diminished the magnitude of window Na+ current (I Na (W)) elicited by short ascending ramp voltage, with unchanged the overall steady-state current-voltage relationship. The decaying time course of I Na during a train of depolarizing pulses arose upon the exposure to RAM. The conditioning train protocol which blocked I Na fitted the recovery time course into two exponential processes and increased the fast and slow time constant of recovery the presence of RAM. In pituitary tumor (GH3) cells, I Na amplitude was also effectively suppressed by the RAM. In addition, GH3-cells exposure to RAM decreased the firing frequency of spontaneous action potentials observed under current-clamp conditions. As a result, the RAM-mediated effect on INa was closely associated with its ability to decrease spontaneous action potentials. Collectively, we found the direct attenuation of I Na and I K (DR) caused by RAM besides the agonistic action on melatonin receptors, which could partially explain its anti-seizure activity.

Function and regulation of serine/threonine phosphatases in the healthy and diseased heart.

Protein phosphorylation is a major control mechanism of a wide range of physiological processes and plays an important role in cardiac pathophysiology. Serine/threonine protein phosphatases control the dephosphorylation of a variety of cardiac proteins, thereby fine-tuning cardiac electrophysiology and function. Specificity of protein phosphatases type-1 and type-2A is achieved by multiprotein complexes that target the catalytic subunits to specific subcellular domains. Here, we describe the composition, regulation and target substrates of serine/threonine phosphatases in the heart. In addition, we provide an overview of pharmacological tools and genetic models to study the role of cardiac phosphatases. Finally, we review the role of protein phosphatases in the diseased heart, particularly in ventricular arrhythmias and atrial fibrillation and discuss their role as potential therapeutic targets.

Inhibitory effects of N-methyl-D-aspartate (NMDA) and α1-adrenergic receptor antagonist ifenprodil on human Kv1.5 channel.

Ifenprodil has been known to reduce cardiac contractility and cerebral vasodilation by antagonizing α1-adrenergic and N-methyl D-aspartate receptor-mediated intracellular signals. This study aimed to investigate the direct effect of ifenprodil on the human voltage-gated Kv1.5 channel (hKv1.5) by using a Xenopus oocyte expression system and a two-microelectrode voltage clamp technique. The amplitudes of hKv1.5 currents, including peak and steady state, were suppressed in a concentration-dependent manner (IC50; 43.1 and 35.5 µM, respectively) after 6 min of ifenprodil treatment. However, these effects were ~ 80% reversed by washout, suggesting that ifenprodil directly inhibited the hKv1.5 independent of membrane receptors or intracellular signals. The inhibition rate of steady state showed voltage dependence, wherein the rates increased according to test voltage depolarization. Ifenprodil reduced the time constants of hKv1.5 inactivation but has higher effects on activation. hKv1.5 inhibition by ifenprodil showed use dependency because the drug more rapidly reduced the current at the higher activation frequencies, and subsequent reduction in frequency after high activation frequency caused a partial channel block relief. Therefore, ifenprodil directly blocked the hKv1.5 in an open state and accelerated the time course of the channel inactivation, which provided a biophysical mechanism for the hKv1.5 blocking effects of ifenprodil.

Inhibitory Effectiveness in Delayed-Rectifier Potassium Current Caused by Vortioxetine, Known to Be a Novel Antidepressant.

Vortioxetine (VOR) is recognized to exert antidepressant actions. However, whether this drug modifies ionic currents in excitable cells remains unclear. The aim of this study was to explore the electrophysiological effects of VOR and other related compounds in pituitary GH3 cells and in Neuro-2a cells. VOR suppressed the delayed-rectifier K+ current (IK(DR)) in a concentration-, time-, and state-dependent manner. Effective IC50 values needed to inhibit peak and sustained IK(DR) were computed to be 31.2 and 8.5 µM, respectively, while the KD value estimated from minimal binding scheme was 7.9 µM. Cell exposure to serotonin (10 µM) alone failed to alter IK(DR), while fluoxetine (10 µM), a compound structurally similar to VOR, mildly suppressed current amplitude. In continued presence of VOR, neither further addition of propranolol nor risperidone reversed VOR-mediated inhibition of IK(DR). Increasing VOR concentration not only depressed IK(DR) conductance but also shifted toward the hyperpolarized potential. As the VOR concentration was raised, the recovery of IK(DR) block became slowed. The IK(DR) activated by a downsloping ramp was suppressed by its presence. The inhibition of IK(DR) by a train pulse was enhanced during exposure to VOR. In Neuro-2a cells, this drug decreased IK(DR). Overall, inhibitory effects of VOR on ionic currents might constitute another underlying mechanism of its actions.

Effective Perturbations by Phenobarbital on INa, IK(erg), IK(M) and IK(DR) during Pulse Train Stimulation in Neuroblastoma Neuro-2a Cells.

Phenobarbital (PHB, Luminal Sodium®) is a medication of the barbiturate and has long been recognized to be an anticonvulsant and a hypnotic because it can facilitate synaptic inhibition in the central nervous system through acting on the γ-aminobutyric acid (GABA) type A (GABAA) receptors. However, to what extent PHB could directly perturb the magnitude and gating of different plasmalemmal ionic currents is not thoroughly explored. In neuroblastoma Neuro-2a cells, we found that PHB effectively suppressed the magnitude of voltage-gated Na+ current (INa) in a concentration-dependent fashion, with an effective IC50 value of 83 µM. The cumulative inhibition of INa, evoked by pulse train stimulation, was enhanced by PHB. However, tefluthrin, an activator of INa, could attenuate PHB-induced reduction in the decaying time constant of INa inhibition evoked by pulse train stimuli. In addition, the erg (ether-à-go-go-related gene)-mediated K+ current (IK(erg)) was also blocked by PHB. The PHB-mediated inhibition on IK(erg) could not be overcome by flumazenil (GABA antagonist) or chlorotoxin (chloride channel blocker). The PHB reduced the recovery of IK(erg) by a two-step voltage protocol with a geometrics-based progression, but it increased the decaying rate of IK(erg), evoked by the envelope-of-tail method. About the M-type K+ currents (IK(M)), PHB caused a reduction of its amplitude, which could not be counteracted by flumazenil or chlorotoxin, and PHB could enhance its cumulative inhibition during pulse train stimulation. Moreover, the magnitude of delayed-rectifier K+ current (IK(DR)) was inhibited by PHB, while the cumulative inhibition of IK(DR) during 10 s of repetitive stimulation was enhanced. Multiple ionic currents during pulse train stimulation were subject to PHB, and neither GABA antagonist nor chloride channel blocker could counteract these PHB-induced reductions. It suggests that these actions might conceivably participate in different functional activities of excitable cells and be independent of GABAA receptors.

The Effectiveness of Isoplumbagin and Plumbagin in Regulating Amplitude, Gating Kinetics, and Voltage-Dependent Hysteresis of erg-mediated K+ Currents.

Isoplumbagin (isoPLB, 5-hydroxy-3-methyl-1,4-naphthoquinone), a naturally occurring quinone, has been observed to exercise anti-inflammatory, antimicrobial, and antineoplastic activities. Notably, whether and how isoPLB, plumbagin (PLB), or other related compounds impact transmembrane ionic currents is not entirely clear. In this study, during GH3-cell exposure to isoPLB, the peak and sustained components of an erg (ether-à-go-go related gene)-mediated K+ current (IK(erg)) evoked with long-lasting-step hyperpolarization were concentration-dependently decreased, with a concomitant increase in the decaying time constant of the deactivating current. The presence of isoPLB led to a differential reduction in the peak and sustained components of deactivating IK(erg) with effective IC50 values of 18.3 and 2.4 µM, respectively, while the KD value according to the minimum binding scheme was estimated to be 2.58 µM. Inhibition by isoPLB of IK(erg) was not reversed by diazoxide; however, further addition of isoPLB, during the continued exposure to 4,4'-dithiopyridine, did not suppress IK(erg) further. The recovery of IK(erg) by a two-step voltage pulse with a geometric progression was slowed in the presence of isoPLB, and the decaying rate of IK(erg) activated by the envelope-of-tail method was increased in its presence. The strength of the IK(erg) hysteresis in response to an inverted isosceles-triangular ramp pulse was diminished by adding isoPLB. A mild inhibition of the delayed-rectifier K+ current (IK(DR)) produced by the presence of isoPLB was seen in GH3 cells, while minimal changes in the magnitude of the voltage-gated Na+ current were demonstrated in its presence. Moreover, the IK(erg) identified in MA-10 Leydig tumor cells was blocked by adding isoPLB. Therefore, the effects of isoPLB or PLB on ionic currents (e.g., IK(erg) and IK(DR)) demonstrated herein would be upstream of our previously reported perturbations on mitochondrial morphogenesis or respiration. Taken together, the perturbations of ionic currents by isoPLB or PLB demonstrated herein are likely to contribute to the underlying mechanism through which they, or other structurally similar compounds, result in adjustments in the functional activities of different neoplastic cells (e.g., GH3 and MA-10 cells), presuming that similar in vivo observations occur.

Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy.

The human ether-á-go-go-related gene (hERG) encodes the pore-forming subunit (Kv11.1), conducting a rapidly delayed rectifier K+ current (I Kr). Reduction of I Kr in pathological cardiac hypertrophy (pCH) contributes to increased susceptibility to arrhythmias. However, practical approaches to prevent I Kr deficiency are lacking. Our study investigated the involvement of ubiquitin ligase Nedd4-2-dependent ubiquitination in I Kr reduction and sought an intervening approach in pCH. Angiotensin II (Ang II) induced a pCH phenotype in guinea pig, accompanied by increased incidences of sudden death and higher susceptibility to arrhythmias. Patch-clamp recordings revealed a significant I Kr reduction in pCH cardiomyocytes. Kv11.1 protein expression was decreased whereas its mRNA level did not change. In addition, Nedd4-2 protein expression was increased in pCH, accompanied by an enhanced Nedd4-2 and Kv11.1 binding detected by immunoprecipitation analysis. Cardiac-specific overexpression of inactive form of Nedd4-2 shortened the prolonged QT interval, reversed I Kr reduction, and decreased susceptibility to arrhythmias. A synthesized peptide containing the PY motif in Kv11.1 C-terminus binding to Nedd4-2 and a cell-penetrating sequence antagonized Nedd4-2-dependent degradation of the channel and increased the surface abundance and function of hERG channel in HEK cells. In addition, in vivo administration of the PY peptide shortened QT interval and action potential duration, and enhanced I Kr in pCH. We conclude that Nedd4-2-dependent ubiquitination is critically involved in I Kr deficiency in pCH. Pharmacological suppression of Nedd4-2 represents a novel approach for antiarrhythmic therapy in pCH.

Acetylcholine Reduces IKr and Prolongs Action Potentials in Human Ventricular Cardiomyocytes.

Vagal nerve stimulation (VNS) has a meaningful basis as a potentially effective treatment for heart failure with reduced ejection fraction. There is an ongoing VNS randomized study, and four studies are completed. However, relatively little is known about the effect of acetylcholine (ACh) on repolarization in human ventricular cardiomyocytes, as well as the effect of ACh on the rapid component of the delayed rectifier K+ current (IKr). Here, we investigated the effect of ACh on the action potential parameters in human ventricular preparations and on IKr in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Using standard microelectrode technique, we demonstrated that ACh (5 µM) significantly increased the action potential duration in human left ventricular myocardial slices. ACh (5 µM) also prolonged repolarization in a human Purkinje fiber and a papillary muscle. Optical mapping revealed that ACh increased the action potential duration in human left ventricular myocardial slices and that the effect was dose-dependent. Perforated patch clamp experiments demonstrated action potential prolongation and a significant decrease in IKr by ACh (5 µM) in hiPSC-CMs. Computer simulations of the electrical activity of a human ventricular cardiomyocyte showed an increase in action potential duration upon implementation of the experimentally observed ACh-induced changes in the fully activated conductance and steady-state activation of IKr. Our findings support the hypothesis that ACh can influence the repolarization in human ventricular cardiomyocytes by at least changes in IKr.

Effective Perturbations of the Amplitude, Gating, and Hysteresis of IK(DR) Caused by PT-2385, an HIF-2α Inhibitor.

PT-2385 is currently regarded as a potent and selective inhibitor of hypoxia-inducible factor-2α (HIF-2α), with potential antineoplastic activity. However, the membrane ion channels changed by this compound are obscure, although it is reasonable to assume that the compound might act on surface membrane before entering the cell´s interior. In this study, we intended to explore whether it and related compounds make any adjustments to the plasmalemmal ionic currents of pituitary tumor (GH3) cells and human 13-06-MG glioma cells. Cell exposure to PT-2385 suppressed the peak or late amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner, with IC50 values of 8.1 or 2.2 µM, respectively, while the KD value in PT-2385-induced shortening in the slow component of IK(DR) inactivation was estimated to be 2.9 µM. The PT-2385-mediated block of IK(DR) in GH3 cells was little-affected by the further application of diazoxide, cilostazol, or sorafenib. Increasing PT-2385 concentrations shifted the steady-state inactivation curve of IK(DR) towards a more hyperpolarized potential, with no change in the gating charge of the current, and also prolonged the time-dependent recovery of the IK(DR) block. The hysteretic strength of IK(DR) elicited by upright or inverted isosceles-triangular ramp voltage was decreased during exposure to PT-2385; meanwhile, the activation energy involved in the gating of IK(DR) elicitation was noticeably raised in its presence. Alternatively, the presence of PT-2385 in human 13-06-MG glioma cells effectively decreased the amplitude of IK(DR). Considering all of the experimental results together, the effects of PT-2385 on ionic currents demonstrated herein could be non-canonical and tend to be upstream of the inhibition of HIF-2α. This action therefore probably contributes to down-streaming mechanisms through the changes that it or other structurally resemblant compounds lead to in the perturbations of the functional activities of pituitary cells or neoplastic astrocytes, in the case that in vivo observations occur.