Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 microM)相似文献   

Prevention of clofilium-induced torsade de pointes by prostaglandin E2 does not involve ATP-dependent K+ channels

Drugs that prolong the QT interval can trigger the life-threatening arrhythmia, torsade de pointes, but there is a poor correlation between the extent of QT prolongation and the occurrence of torsade de pointes. The clinical status of a patient may modify the arrhythmogenicity of drugs; thus, we have investigated whether a mediator of fever and inflammation, prostaglandin E(2), alters the proarrhythmic effects of clofilium. In pentobarbitone-anaesthetized, open-chest, alpha-adrenoceptor-stimulated rabbits, prostaglandin E(2) 0.28, 0.84 and 2.80 nmol kg(-1) min(-1), infused into the left ventricle, reduced the incidence of torsade de pointes from 50% in controls to 20%, 20% and 0%, respectively (n=10 per group). Pretreatment with glibenclamide (10 micromol kg(-1)) did not alter the antiarrhythmic effect of prostaglandin E(2) (2.80 nmol kg(-1) min(-1)). These results indicate that prostaglandin E(2) prevents drug-induced torsade de pointes and that this action of prostaglandin E(2) is not mediated via opening of ATP-dependent K(+) channels (K(ATP)).     

Effects of U-37883A on intracellular Ca2+ -activated large-conductance K+ channels in pig proximal urethral myocytes

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.     

Effects of ginsenoside on G protein-coupled inwardly rectifying K+ channel activity expressed in Xenopus oocytes

Recently, we provided evidence that ginsenoside, the active component of Panax ginseng, uses the pertussis toxin-insensitive Gα_q/11-phospholipase C-β3 signal transduction pathway to increase Ca²⁺-activated Cl⁻ currents in the Xenopus oocyte. Other investigators have shown that stimulation of receptors linked to the Gα_q-phospholipase C pathway inhibits the activity of G protein-coupled inwardly rectifying K⁺ (GIRK) channels. In the present study, we sought to determine whether ginsenoside influenced the activity of GIRK 1 and GIRK 4 (GIRK 1/4) channels expressed in the Xenopus oocyte, and if so, the underlying signal transduction mechanism. In oocytes injected with GIRK 1/4 channel cRNA, bath-applied ginsenoside inhibited the high K⁺ solution-elicited GIRK current (EC₅₀: 4.9±4.3 μg/ml). Pretreatment of the oocyte with pertussis toxin reduced the high K⁺ solution-elicited GIRK current by 49%, but it did not alter the inhibitory effect of ginsenoside on the GIRK current. Prior intraoocyte injection of cRNA(s) coding Gα_q, Gα₁₁ or Gα_q/Gα₁₁, but not Gα_i2 or Gα_oA, attenuated the inhibitory ginsenoside effect. Injection of cRNAs coding Gβ₁γ₂ also attenuated the ginsenoside effect. Preincubation of GIRK channel-expressing oocytes with phospholipase C inhibitor, {1-[6-((17b-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione} (U73122), or protein kinase C inhibitor, staurosporine or chelerythrine, blocked the inhibitory ginsenoside effect on the GIRK current. Intraoocyte injection of bis (o-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid (BAPTA), a free Ca²⁺ chelator, had no significant effect on the action of ginsenoside. Taken together, these results suggest that ginsenoside inhibits the activity of the GIRK 1/4 channel expressed in the Xenopus oocyte through a pertussis toxin-insensitive and Gα_q/11-, phospholipase C- and protein kinase C-mediated signal transduction pathway.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

Differential blockade of neuronal voltage-gated Na(+) and K(+) channels by antidepressant drugs

The effects of a range of antidepressants were investigated on neuronal voltage-gated Na(+) and K(+) channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na(+) uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na(+) channel. Imipramine also produced a differential action on macroscopic Na(+) and K(+) channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na(+) channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na(+) channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na(+) channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K(+) currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.     

Effects of fluoroquinolones on insulin secretion and beta-cell ATP-sensitive K+ channels

Although fluoroquinolones are used widely in the treatment of various infectious diseases, some of the drugs are known to cause hypoglycemia as a side-effect. We have investigated the effects of three fluoroquinolone derivatives, levofloxacin, gatifloxacin, and temafloxacin, on insulin secretion and pancreatic beta-cell ATP-sensitive K(+) channel (K(ATP) channel) activity. While levofloxacin had only a small effect on insulin secretion and K(ATP) currents, gatifloxacin and temafloxacin stimulated insulin secretion and inhibited K(ATP) channel currents in a dose-dependent manner. We also determined the site of action of gatifloxacin and temafloxacin on the K(ATP) channel. In a reconstituted system, gatifloxacin and temafloxacin inhibited Kir6.2 Delta C26 channels, which function in the absence of the SUR subunit, indicating direct action of the drugs on the Kir6.2 subunits. These results suggest that stimulation of insulin secretion by inhibition of pancreatic beta-cell K(ATP) channels underlies the hypoglycemia caused by certain fluoroquinolones.     

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein

Summary The resting membrane potential of smooth muscle cells of the rabbit portal vein was –51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to –62.3 mV (10 M) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 M) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of –40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was –82 mV and this value was close to the equilibrium potential for K⁺ (–80 mV) in the present ionic conditions, suggesting that LP-805 activated the K⁺ channel. Generation of both the hyperpolarization and the outward c urrent by LP-805 was inhibited by glibenclamide ( 1 M). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K⁺ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K⁺ channel current (15 pS) without change in the activity of the maxi-K⁺ channel. The maxi-K⁺ channel was sensitive to charybdotoxin (0.1 M) and to intracellular Ca²⁺ ([Ca²⁺]_i) concentration. The 15 pS channel was insensitive to [Ca²⁺]_i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 M) inhibited the 15 pS K⁺ channel activated by LP-805. These actions of LP-805 on the maxi-K⁺ and 15 pS K⁺ channels are the same as those previously observed for nicorandil and pinacidil. Thus, LP-805 is a K⁺ channel opener with a chemical structure different from those of the known openers. Correspondence to M. Kamouchi at the above address     

Toxin modulators and blockers of hERG K channels

The K⁺ channel encoded by the Ether-á-go-go-Related Gene (ERG) is expressed in different tissues of different animal species. There are at least three subtypes of this channel, being the sub-type 1 (ERG1) crucial in the repolarization phase of the cardiac action potential. Mutations in this gene can affect the properties of the channel producing the type II long QT syndrome (LQTS2) and many drugs are also known to affect this channel with a similar side effect. Various scorpion, spider and sea anemone toxins affect the ERG currents by blocking the ion-conducting pore from the external side or by modulating channel gating through binding to the voltage-sensor domain. By doing so, these toxins become very useful tools for better understanding the structural and functional characteristics of these ion channels. This review discusses the interaction between the ERG channels and the peptides isolated from venoms of these animals. Special emphasis is placed on scorpion toxins, although the effects of several spider venom toxins and anemone toxins will be also revised.     

Sustained K+ Outward Currents are Sensitive to Intracellular Heteropodatoxin2 in CA1 Neurons of Organotypic Cultured Hippocampi of Rats

Blocking or regulating K⁺ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent K⁺ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electrophysiologically if heteropodatoxin2 (HpTX₂), known as one of Kv4-specific toxins, might be effective on various K⁺ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total K⁺ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of HpTX₂ weakly but significantly reduced transient currents. However, when HpTX₂ was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of HpTX₂ effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic HpTX₂ is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of HpTX₂ inside and outside of neurons are very efficient to selectively reduce specific K⁺ outward currents.     

Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel opening drugs minoxidil sulphate and cromakalim, on ⁴²K⁺ and ⁸⁶Rb⁺ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD₂ values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of ⁴²K⁺ and ⁸⁶Rb⁺ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated ⁸⁶Rb⁺/⁴²K⁺ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the ⁸⁶Rb⁺ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux in a concentration-dependent manner with IC₅₀ values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K⁺ channel opener, and may act to open a homogeneous population of K⁺ channels. In contrast, the actions of cromakalim (1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K⁺ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K⁺ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide. Send offprint requests to U. Quasi at the above address     

Block of hERG K+ Channel by Classic Histamine H1 Receptor Antagonist Chlorpheniramine

Chlorpheniramine is a potent first-generation histamine H₁ receptor antagonist that can increase action potential duration and induce QT prolongation in several animal models. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of leading causes of acquired long QT syndrome, we investigated the acute effects of chlorpheniramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of chlorpheniramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Chlorpheniramine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC₅₀ of chlorpheniramine-dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. Chlorpheniramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that the H₁ antihistamine, chlorpheniramine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Antinociceptive action of myricitrin: involvement of the K+ and Ca2+ channels

The present study was designed to investigate the mechanisms involved in the antinociception afforded by myricitrin in chemical models of nociception in mice. Myricitrin given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) route produced dose-related antinociception when evaluated against acetic acid-induced visceral pain in mice. In addition, the intraperitoneal administration of myricitrin caused significant inhibition of biting behaviour induced by i.t. injection of glutamate, substance P, capsaicin, interleukin 1 β (IL-1β) and tumor necrosis factor-α (TNF-α). The antinociception caused by myricitrin in the acetic acid test was fully prevented by i.t. pre-treatment with pertussis toxin, a Gi/o protein inactivator, and by i.c.v. injection of calcium chloride (CaCl₂). In addition, the i.t. pre-treatment of mice with apamin, a blocker of small (or low)-conductance calcium-gated K⁺ channels and tetraethylammonium, a blocker of voltage-gated K⁺ channels significantly reversed the antinociception induced by myricitrin. The charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K⁺ channels and glibenclamide, a blocker of the ATP-gated K⁺ channels had no effect on myricitrin-induced antinociception. Calcium uptake analysis revealed that myricitrin inhibited ⁴⁵Ca²⁺ influx under a K⁺-induced depolarization condition. However, calcium movement was modified in a non-depolarizing condition only when the highest concentration of myricitrin was used. In summary, our findings indicate that myricitrin produces consistent antinociception in chemical models of nociception in mice. These results clearly demonstrate an involvement of the Gi/o protein dependent mechanism on antinociception caused by myricitrin. The opening of voltage- and small-conductance calcium-gated K⁺ channels and the reduction of calcium influx led to the antinociceptive of myricitrin.     

Insulin induced translocation of Na^＋/K^＋-ATPase is decreased in the heart of streptozotocin diabetic rats

Aim:

To investigate the effect of acute insulin administration on the subcellular localization of Na⁺/K⁺-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.

Methods:

Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na⁺/K⁺-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.

Results:

In control rat heart muscle α1 isoform of Na⁺/K⁺ ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P<0.05) in plasma membrane and α2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2- but not α1-subunits was detected. α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and β1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.

Conclusion:

This study demonstrate that insulin influences the plasma membrane localization of Na⁺/K⁺-ATPase isoforms in the heart. α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na⁺/K⁺-ATPase α1- and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.     

Selective modulation of the ATP-sensitive K+ channel by nicorandil in guinea-pig cardiac cell membrane

Summary Effects of a vasodilator, nicorandil (2-nicotinamidoethyl nitrate) on four kinds for cardiac K⁺ channels were investigated in guinea pig ventricular and atrial cells using inside-out patch recording combined with oilgate concentration jump method.Nicorandil of 300 mol/l failed to affect the inward-rectifier K⁺ channel and the Na⁺-activated K⁺ channel. The open probability of the muscarinic K⁺ channel, when activated by the application of GTP, was not changed by the drug. Nicorandil selectively increased the open probability of the ATP-sensitive K⁺ channel that was partly suppressed by intracellular ATP. The median effective concentration (EC50) of nicorandil was 74 mol/l and Hill coefficient was 1.32 in the concentration-open probability relationship. The closing rate of the K⁺ channel by ATP was markedly delayed by the drug, whereas the open rate on removal of ATP was scarcely affected. Nicorandil had only little effect on this channel after run-down. It was concluded that nicorandil selectively activates the ATP-sensitive K⁺ channel mainly by modulating the ATP-dependent gate.Send offprint requests to M. Takano at the above address     

Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells

1. The action of the anticonvulsant drug phenytoin on K⁺ currents was investigated in neuroblastoma cells by whole-cell voltage-clamp recording.
2. Neuroblastoma cells expressed an outward K⁺ current with a voltage- and time-dependence which resembled the delayed-rectifier K⁺ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n=65). Inhibition was concentration-dependent with a half-maximal inhibitory concentration of 30.9±0.8 μM.
3. The K⁺ current inhibition by phenytoin was voltage-dependent with block by phenytoin being relieved by depolarization.
4. The times taken to reach steady-state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K⁺ current nor the K⁺ channel availability were significantly altered by the blocking drug. A use-dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.
5. These results suggest that phenytoin affects K⁺ currents and that this effect might lead to a reduction in neuronal excitability.

     

Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

Background and Purpose

Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I_Kr currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K⁺ channels.

Experimental Approach

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

Key Results

Zolpidem caused acute hERG channel blockade in oocytes (IC₅₀ = 61.5 μM) and in HEK 293 cells (IC₅₀ = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I–V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes.

Conclusions and Implications

Zolpidem inhibits cardiac hERG K⁺ channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.