Characterization of aconitine-induced block of delayed rectifier K+ current in differentiated NG108-15 neuronal cells

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

L-364,373 fails to activate the slow delayed rectifier K+ current in canine ventricular cardiomyocytes

Activators of the slow delayed rectifier K⁺ current (I_Ks) are promising tools to suppress ventricular arrhythmias originating from prolongation of action potentials. A recently synthesized compound, L-364,373, was shown to activate I_Ks in ventricular cells isolated from guinea pigs and rabbits. Due to the interspecies differences known to exist in the properties of the delayed rectifier K⁺ currents, the effect of L-364,373 on I_Ks was studied and compared with that of another I_Ks activator mefenamic acid in canine ventricular myocytes. Mefenamic acid (100 μM) significantly increased the amplitude of the fully activated I_Ks current, as well as the I_Ks current tails, by shifting the voltage dependence of its activation towards negative voltages and increased the time constant for deactivation. In contrast, L-364,373, up to concentrations of 3 μM, failed to augment I_Ks at any membrane potential studied, but slightly increased the time constant of deactivation. It is concluded that human studies are required to evaluate the therapeutically beneficial effects of I_Ks activators. Rodent cardiac tissues are not suitable for this purpose.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

Inhibitory action of ICI-182,780, an estrogen receptor antagonist, on BK(Ca) channel activity in cultured endothelial cells of human coronary artery

ICI-182,780 is known to be a selective inhibitor of the intracellular estrogen receptors. The effect of ICI-182,780 on ion currents was studied in cultured endothelial cells of human coronary artery. In whole-cell current recordings, ICI-182,780 reversibly decreased the amplitude of K(+) outward currents. The decrease in outward current caused by ICI-182,780 could be counteracted by further application of magnolol or nordihydroguaiaretic acid, yet not by 17beta-estradiol. Under current-clamp condition, ICI-182,780 (3microM) depolarized the membrane potentials of the cells, and magnolol (10 microM) or nordihydroguaiaretic acid (10 microM) reversed ICI-182,780-induced depolarization. In inside-out patches, ICI-182,780 added to the bath did not alter single-channel conductance of large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels), but decreased their open probability. ICI-182,780 reduced channel activity in a concentration-dependent manner with an IC(50) value of 3 microM. After BK(Ca) channel activity was suppressed by 2-methoxyestradiol (3 microM), subsequent application of ICI-182,780 (3 microM) did not further reduce the channel activity. The application of ICI-182,780 shifted the activation curve of BK(Ca) channels to positive potentials. Its decrease in the open probability primarily involved a reduction in channel open duration. ICI-182,780 also suppressed the proliferation of these endothelial cells with an IC(50) value of 2 microM. However, in coronary smooth muscle cells, a bell-shaped concentration-response curve for the ICI-182,780 effect on BK(Ca) channel activity was observed. This study provides evidence that ICI-182,780 can inhibit BK(Ca) channels in vascular endothelial cells in a mechanism unlikely to be linked to its anti-estrogen activity. The inhibitory effects on these channels may partly contribute to the underlying mechanisms by which ICI-182,780 affects endothelial function.     

Activation of K+ channel by 1-EBIO rescues the head and neck squamous cell carcinoma cells from Ca2+ ionophore-induced cell death

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca²⁺ ([Ca²⁺]_i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca²⁺-activated K⁺ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 µM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca²⁺-activated Cl^- channels (Ano-1) and Ca²⁺-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K⁺ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca²⁺ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.     

Inhibition of ATP-sensitive K(+)-channels by a sulfonylurea analogue with a phosphate group

Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K(+)-channels in the pancreatic beta-cells. These channels are composed of two proteins, a pore-forming subunit (K(IR)6.2 in the case of beta-cells) and a regulatory subunit, the sulfonylurea receptor (SUR). In the present study we characterised the interaction with SURs of the new sulfonylurea analogues 5-chloro-N-[2-(4-hydroxyphenyl)ethyl]-2-methoxybenzamide (compound I) and (4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl)phosphate (compound II). Compounds I and II differ from the sulfonylurea analogue meglitinide only in so far as the carboxylic group of meglitinide is replaced by a hydroxyl group or a phosphate group, respectively. The binding affinities of compound II for the SUR subtypes SUR1 (identified in beta-cells) and SUR2A (identified in heart and skeletal muscle) were higher by 55 or 21-fold, respectively, than the corresponding affinities for compound I. In inside-out patch-clamp experiments compound II inhibited ATP-sensitive K(+)-channels of the SUR1/K(IR)6.2-type (characteristic of beta-cells) with an IC(50) value of 0.16 microM which is 6-fold lower than the corresponding value for meglitinide. These findings strongly support the conclusion that the interaction of sulfonylureas and acidic analogues with SURs is favoured by the anionic group of these drugs and that a phosphate group allows more efficient ligand interaction with SUR1 than a carboxylic group.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Imipramine inhibits A-type delayed rectifier and ATP-sensitive K+ currents independent of G-protein and protein kinase C in murine proximal colonic myocytes

The effects of imipramine on A-type delayed rectifier K+ currents and ATP-sensitive K+ (KATP) currents were studied in isolated murine proximal colonic myocytes using the whole-cell patch-clamp technique. Depolarizing test pulses between -80 mV and +30 mV with 10 mV increments from the holding potential of -80 mV activated voltage-dependent outward K+ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier K+ currents was reduced by external application of imipramine. The half-inactivation potential and the half-recovery time of A-type delayed rectifier K+ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of -80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited KATP currents. The inhibitory effects of imipramine in A-type delayed rectifier K+ currents and KATP currents were not changed by guanosine 5-O-(2-thiodiphosphate) (GDPbetaS) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier K+ currents and KATP currents in a manner independent of G-protein and protein kinase C.     

Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid,on ion currents in NG108-15 neuronal cells

The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.     

High potency inhibition of hERG potassium channels by the sodium-calcium exchange inhibitor KB-R7943

BACKGROUND AND PURPOSE

KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium–calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K⁺ channels that underpin the cardiac rapid delayed rectifier potassium current, I_Kr.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp measurements were made of hERG current (I_hERG) carried by wild-type or mutant hERG channels and of native rabbit ventricular I_Kr. Docking simulations utilized a hERG homology model built on a MthK-based template.

KEY RESULTS

KB-R7943 inhibited both I_hERG and native I_Kr rapidly on membrane depolarization with IC₅₀ values of ∼89 and ∼120 nM, respectively, for current tails at −40 mV following depolarizing voltage commands to +20 mV. Marked I_hERG inhibition also occurred under ventricular action potential voltage clamp. I_hERG inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound''s nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited I_hERG, but with a markedly reduced potency.

CONCLUSIONS AND IMPLICATIONS

KB-R7943 inhibits I_hERG/I_Kr with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG.     

The anti-protozoal drug pentamidine blocks KIR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel

Background and purpose:

Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (I_K1), we examined the action and mechanism of pentamidine-mediated I_K1 block.

Experimental approach:

Patch clamp measurements of I_K1 were made on cultured adult canine ventricular cardiomyocytes, K_IR2.1-HEK293 cells and K_IR2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of K_IR2.1 channels was studied by molecular modelling.

Key results:

Pentamidine application (24 h) decreased I_K1 in cultured canine cardiomyocytes and K_IR2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited I_K1 in K_IR2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of I_K1 was acute (IC₅₀= 0.17 µM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of K_IR2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of I_K1. Block was independent of the presence of spermine. K_IR2.2, and K_IR2.3 based I_K1 was also sensitive to pentamidine blockade.

Conclusions and implications:

Pentamidine inhibits cardiac I_K1 by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific I_K1 blocking compounds.     

Modulation of the delayed K+ current by histamine in guinea pig ventricular myocytes

Summary The effects of histamine on delayed K⁺ current (I_K) were investigated in patch-clamped single guinea pig ventricular myocytes. Histamine increased I_K with a maximal fractional response of 2.7 and a k_d of 9.4 × 10^–7 mol/l. At a concentration of 10^–8 mol/l, histamine did not increase I_K significantly, but increased I_Ca by 52% ± 12%. The voltage-dependence of I_K activation, the reversal potential and the time course of the I_K tail decay were not changed by histamine. Under pretreatment with 10^–4 mol/l of ranitidine, neither histamine (10^–6 mol/l) nor 2-pyridylethylamine (10^–4 mol/l) caused any sizable increase in I_K. When the cell was pretreated with a saturating dose of isoproterenol (10^–6 mol/l), histamine did not additively enhance I_K. The I_K enhancement by 3 × 10^–7 mol/l histamine was partially antagonized by concurrent exposure to 5 × 10^–6 mol/l carbachol. Whereas, use of a higher concentration of histamine (10^–6 mol/l) obscured the inhibitory effect of carbachol. It is concluded that histaminergic action of I_K is attributed exclusively to H₂ receptor-mediated reactions involving G_s protein and adenylate cyclase. Send offprint requests to Y. Habuchi at the above address     

Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes

The mechanism of drug-induced inhibition of the transient outward current, I_to, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. I_to was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from –50 mV up to +40 mV. At +40 mV, I_to peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, _fast = 17 ms and _slow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed I_to at the end of the 300 ms depolarizing voltage clamp step; the inactivation of I_to was accelerated by all drugs, whereas peak I_to was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of I_to. Verapamil, quinidine sulfate and nifedipine exerted a block of I_to. increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 mol/each) was appropriately described by monoexponential functions with time constants _on = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at –50 mV was assessed by comparison of the recovery process of peak I_to from inactivation with or without drugs. _off amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible. 4-Aminopyridine preferentially depressed peak I_to in a concentration-dependent manner, whereas I_to at the end of the 300 ms depolarizing voltage step remained unaffected. The block of I_to by 4-aminopyridine (3 mmol/l) decreased during the voltage step from –50 mV to +40 mV. Relief from block was described by _off = 30.4 ms. The efficacy of 4-aminopyridine was diminished at short and enhanced at long pulse intervals (reverse use-dependence). The time course of 4-aminopyridine-induced block of Ito was described by _on = 1561 ms. Phenylephrine (30 mol/l),papaverine (30 mol/I) and tetraethylammonium chloride (5 mmol/l) reduced I_to at the peak and at the end of the 300 ms depolarizing voltage step in a time-independent manner. It is concluded that verapamil, quinidine sulfate and nifedipine bind to the I_to channel in the open state at positive membrane potentials. In contrast, 4-aminopyridine obviously binds to the channel in the closed state at negative membrane potentials. Phenylephrine, papaverine and tetraethylammonium chloride seem to block I_to independent of the channel state. Correspondence to: H. Nawrath at the above address     

Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na⁺,K⁺-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na⁺,K⁺-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na⁺,K⁺-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (k_obs). From the representation of k_obs versus PLTX concentration, the kinetic equilibrium dissociation constant (K_D) for the PLTX-Na⁺,K⁺-ATPase association can be calculated. The value of this constant is K_D = 6.38 × 10⁻⁷ ± 6.67 × 10⁻⁸ M PLTX. In this way the PLTX-Na⁺,K⁺-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.     

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.     

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   

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.