Voltage Of Half-maximal Inactivation Research Articles

The fungicide tebuconazole modulates the sodium current of human NaV1.5 channels expressed in HEK293cells.

The fungicide Tebuconazole is a widely used pesticide in agriculture and may cause cardiotoxicity. In our present investigation the effect of Tebuconazole on the sodium current (INa) of human cardiac sodium channels (NaV1.5) was studied using a heterologous expression system and whole-cell patch-clamp techniques. Tebuconazole reduced the amplitude of the peak INa in a concentration- and voltage-dependent manner. At the holding potential of -120 mV the IC50 was estimated at 204.1±34.3μM, while at -80 mV the IC50 was 0.3±0.1μM. The effect of the fungicide is more pronounced at more depolarized potentials, indicating a state-dependent interaction. Tebuconazole caused a negative shift in the half-maximal inactivation voltage and delayed recovery from fast inactivation of INa. Also, it enhanced closed-state inactivation, exhibited use-dependent block in a voltage-dependent manner. Furthermore, Tebuconazole reduced the increase in late sodium current induced by the pyrethroid insecticide β-Cyfluthrin. These results suggest that Tebuconazole can interact with NaV1.5 channels and modulate INa. The observed effects may lead to decreased cardiac excitability through reduced INa availability, which could be a new mechanism of cardiotoxicity to be attributed to the fungicide.

The volatile anesthetic isoflurane differentially inhibits voltage-gated sodium channel currents between pyramidal and parvalbumin neurons in the prefrontal cortex.

How volatile anesthetics work remains poorly understood. Modulations of synaptic neurotransmission are the direct cellular mechanisms of volatile anesthetics in the central nervous system. Volatile anesthetics such as isoflurane may reduce neuronal interaction by differentially inhibiting neurotransmission between GABAergic and glutamatergic synapses. Presynaptic voltage-dependent sodium channels (Nav), which are strictly coupled with synaptic vesicle exocytosis, are inhibited by volatile anesthetics and may contribute to the selectivity of isoflurane between GABAergic and glutamatergic synapses. However, it is still unknown how isoflurane at clinical concentrations differentially modulates Nav currents between excitatory and inhibitory neurons at the tissue level. In this study, an electrophysiological recording was applied in cortex slices to investigate the effects of isoflurane on Nav between parvalbumin (PV+) and pyramidal neurons in PV-cre-tdTomato and/or vglut2-cre-tdTomato mice. Isoflurane at clinically relevant concentrations produced a hyperpolarizing shift in the voltage-dependent inactivation and slowed the recovery time from the fast inactivation in both cellular subtypes. Since the voltage of half-maximal inactivation was significantly depolarized in PV+ neurons compared to that of pyramidal neurons, isoflurane inhibited the peak Nav currents in pyramidal neurons more potently than those of PV+ neurons (35.95 ± 13.32% vs. 19.24 ± 16.04%, P = 0.036 by the Mann-Whitney test). Isoflurane differentially inhibits Nav currents between pyramidal and PV+ neurons in the prefrontal cortex, which may contribute to the preferential suppression of glutamate release over GABA release, resulting in the net depression of excitatory-inhibitory circuits in the prefrontal cortex.

Changes in Resurgent Sodium Current Contribute to the Hyperexcitability of Muscles in Patients with Paramyotonia Congenita.

Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Nav1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Nav1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Nav1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype.

Inhibition of voltage-gated Na+ currents by eleclazine in rat atrial and ventricular myocytes

BackgroundAtrial-ventricular differences in voltage-gated Na+ currents might be exploited for atrial-selective antiarrhythmic drug action for the suppression of atrial fibrillation without risk of ventricular tachyarrhythmia. Eleclazine (GS-6615) is a putative antiarrhythmic drug with properties similar to the prototypical atrial-selective Na+ channel blocker ranolazine that has been shown to be safe and well tolerated in patients.ObjectiveThe present study investigated atrial-ventricular differences in the biophysical properties and inhibition by eleclazine of voltage-gated Na+ currents.MethodsThe fast and late components of whole-cell voltage-gated Na+ currents (respectively, INa and INaL) were recorded at room temperature (∼22°C) from rat isolated atrial and ventricular myocytes.ResultsAtrial INa activated at command potentials ∼5.5 mV more negative and inactivated at conditioning potentials ∼7 mV more negative than ventricular INa. There was no difference between atrial and ventricular myocytes in the eleclazine inhibition of INaL activated by 3 nM ATX-II (IC50s ∼200 nM). Eleclazine (10 μM) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated state block. Eleclazine produced voltage-dependent instantaneous inhibition in atrial and ventricular myocytes; it caused a negative shift in voltage of half-maximal inactivation and slowed the recovery of INa from inactivation in both cell types.ConclusionsDifferences exist between rat atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation/inactivation in atrial myocytes underlies differences between the 2 cell types in the voltage dependence of instantaneous inhibition by eleclazine. Eleclazine warrants further investigation as an atrial-selective antiarrhythmic drug.

Eleclazine Inhibition of Voltage-gated Sodium Currents in Rat Atrial and Ventricular Myocytes

Atrial-ventricular differences in voltage-gated Na+ currents might be exploited for atrial-selective antiarrhythmic drug action for the suppression of atrial fibrillation without risk of ventricular tachyarrhythmia side-effects. Eleclazine (GS-6615) is a putative antiarrhythmic drug that has been shown to be safe and well-tolerated in patients and has properties similar to the prototypical atrial-selective Na+ channel blocker, ranolazine.Atrial-ventricular differences in the biophysical properties of voltage-gated Na+ currents and their inhibition by eleclazine were investigated.The fast and late components of whole-cell voltage-gated Na+ currents (respectively, INa & INaL) were recorded at room temperature (∼22 °C) from rat isolated atrial and ventricular myocytes. Atrial INa activated at command potentials ∼5.5 mV more negative and inactivated at conditioning potentials ∼7 mV more negative than ventricular INa. Under control conditions, INaL density was ∼2-fold greater in atrial than in ventricular myocytes. There was no difference between the two cell types in block of INaL by eleclazine (IC50s ∼200 nM) in the presence of the INaL activator, ATX-II (3 nM). 10 μM eleclazine inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated state block. Eleclazine produced an instantaneous inhibition of INa in atrial but not ventricular myocytes; the drug caused a negative shift in voltage of half-maximal inactivation and slowed the recovery of INa from inactivation in both cell types. Thus, differences exist between rat atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage-dependence of INa activation/inactivation underlies the instantaneous block by eleclazine in atrial myocytes. Eleclazine warrants further investigation as an atrial-selective antiarrhythmic drug.

Cytoskeleton disruption affects Kv2.1 channel function and its modulation by PIP2.

Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.

Atrial-ventricular differences in rabbit cardiac voltage-gated Na+ currents: Basis for atrial-selective block by ranolazine

BackgroundClass 1 antiarrhythmic drugs are highly effective in restoring and maintaining sinus rhythm in atrial fibrillation patients but carry a risk of ventricular tachyarrhythmia. The antianginal agent ranolazine is a prototypic atrial-selective voltage-gated Na+ channel blocker but the mechanisms underlying its atrial-selective action remain unclear.ObjectiveThe present study examined the mechanisms underlying the atrial-selective action of ranolazine.MethodsWhole-cell voltage-gated Na+ currents (INa) were recorded at room temperature (∼22°C) from rabbit isolated left atrial and right ventricular myocytes.ResultsINa conductance density was ∼1.8-fold greater in atrial than in ventricular cells. Atrial INa was activated at command potentials ∼7 mV more negative and inactivated at conditioning potentials ∼11 mV more negative than ventricular INa. The onset of inactivation of INa was faster in atrial cells than in ventricular myocytes. Ranolazine (30 μM) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated/inactivated state block. Ranolazine caused a significantly greater negative shift in voltage of half-maximal inactivation in atrial cells than in ventricular cells, the recovery from inactivation of INa was slowed by ranolazine to a greater extent in atrial myocytes than in ventricular cells, and ranolazine produced an instantaneous block that showed marked voltage dependence in atrial cells.ConclusionDifferences exist between rabbit atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation and inactivation, together with trapping of the drug in the inactivated channel, underlies an atrial-selective action of ranolazine.

Temperature-dependence of L-type Ca(2+) current in ventricular cardiomyocytes of the Alaska blackfish (Dallia pectoralis).

To lend insight into the overwintering strategy of the Alaska blackfish (Dallia pectoralis), we acclimated fish to 15 or 5 °C and then utilized whole-cell patch clamp to characterize the effects of thermal acclimation and acute temperature change on the density and kinetics of ventricular L-type Ca(2+) current (I Ca). Peak I Ca density at 5 °C (-1.1 ± 0.1 pA pF(-1)) was 1/8th that at 15 °C (-8.8 ± 0.6 pA pF(-1)). However, alterations of the Ca(2+)- and voltage-dependent inactivation properties of L-type Ca(2+) channels partially compensated against the decrease. The time constant tau (τ) for the kinetics of inactivation of I Ca was ~4.5 times greater at 5 °C than at 15 °C, and the voltage for half-maximal inactivation was shifted from -23.3 ± 1.0 mV at 15 °C to -19.8 ± 1.2 mV at 5 °C. These modifications increase the open probability of the channel and culminate in an approximate doubling of the L-type Ca(2+) window current, which contributes to approximately 15% of the maximal Ca(2+) conductance at 5 °C. Consequently, the charge density of I Ca (Q Ca) and the total Ca(2+) transferred through the L-type Ca(2+) channels (Δ[Ca(2+)]) were not as severely reduced at 5 °C as compared to peak I Ca density. In combination, the results suggest that while the Alaska blackfish substantially down-regulates I Ca with acclimation to low temperature, there is sufficient compensation in the kinetics of the L-type Ca(2+) channel to support the level of cardiac performance required for the fish to remain active throughout the winter.

Gastric emptying and Ca2+ and K+ channels of circular smooth muscle cells in diet‐induced obese prone and resistant rats

Accelerated gastric emptying that precipitates hunger and frequent eating could be a potential factor in the development of obesity. The aim of this study was to study gastric emptying in diet-induced obese-prone (DIO-P) and DIO-resistant (DIO-R) rats and explore possible differences in electrical properties of calcium (Ca(2+) ) and potassium (K(+) ) channels of antral circular smooth muscle cells (SMCs). Whole-cell patch-clamp technique was used to measure Ca(2+) and K(+) currents in single SMCs. Gastric emptying was evaluated 90 min after the ingestion of a solid meal. Solid gastric emptying in the DIO-P rats was significantly faster compared with that in the DIO-R rats. The peak amplitude of L-type Ca(2+) current (IBa,L ) at 10 mV in DIO-P rats was greater than that in DIO-R rats without alternation of the current-voltage curve and voltage-dependent activation and inactivation. The half-maximal inactivation voltage of transient outward K(+) current (IKto ) was more depolarized (∼4 mV) in DIO-P rats compared with that in DIO-R rats. No difference was found in the current density or recovery kinetics of IKto between two groups. The current density of delayed rectifier K(+) current (IKdr ), which was sensitive to tetraethylammonium chloride but not 4-aminopyridine, was lower in DIO-P rats than that in DIO-R rats. The accelerated gastric emptying in DIO-P rats might be attributed to a higher density of IBa,L , depolarizing shift of inactivation curve of IKto and lower density of IKdr observed in the antral SMCs of DIO-P rats.

Combined Stopped-Flow and Electrophysiological Experiments Suggest Direct Sodium Channel Inhibition by Model Fluorobenzene Anesthetics

General anesthetics are often proposed to affect membrane properties through interactions with the lipid bilayer. Though recent studies have shown that inhaled anesthetics can interact more specifically with certain membrane proteins such as ion channels. For example, volatile anesthetics inhibit voltage-gated sodium channels to reduce neurotransmitter release. Fluorobenzenes (FBs), once considered for clinical use, were abandoned due to their flammability and toxicity but are still valuable model anesthetics for investigating the molecular mechanisms of anesthetics. We examined the properties of four FB compounds, 1,2-DiFB, 1,4-DiFB, 1,3,5-TriFB and HexaFB on lipid bilayer and sodium channel function at equipotent clinically relevant concentrations. Effects on lipid bilayer properties were tested using a gramicidin channel based stopped-flow fluorescence assay for lipid bilayer perturbation; effects on sodium channel function were tested using whole-cell voltage-clamp electrophysiology on neuronal cells (ND7/23). The stopped-flow results showed that all four FBs minimally affected lipid bilayer properties, whereas the sodium channels were strongly inhibited by all four anesthetics. Inhibition of peak sodium current was voltage-dependent as a pre-pulse to a voltage at which half the channels were in the fast inactivated state (V1/2) revealed strong inhibition compared to a pre-pulse to a voltage at which the majority of the channels were in the resting state (V0). The FBs produce a left-shift in the voltage of half-maximal inactivation (V1/2, also known as h∞ or availability), with 1,2-DiFB showing the greatest and HexaFB the least shift; these changes are comparable to those observed with modern inhaled anesthetics such as isoflurane. Together these results suggest that these compounds alter sodium channel function through direct interactions with the channels, though we cannot exclude that membrane effects may become involved at high, supra-pharmacological concentrations.

Functional Analysis of Missense Mutations in Kv8.2 Causing Cone Dystrophy with Supernormal Rod Electroretinogram

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.

Gamma1 Subunit Renders Cav1.2 Channels Dependent on Cell Cycle

Auxiliary γ subunits are known to enhance inactivation of L-type calcium channels. When co-expressed in tsA-201 cells with α1C and β2a subunits, the γ1 subunit shifts the voltage-dependence of inactivation by about −20 mV. The voltage of half-maximal inactivation, V1/2, was determined with 5 s long conditioning pre-pulses. On average, addition of γ1 to α1C/β2a channels changed V1/2 from −24±5 mV (n=30) to −44±12 mV (n=92). We noticed that V1/2, but not the steepness of the voltage-dependence of inactivation, varies greatly in cells with γ1 and set up to find the cause of the cell-to-cell variability. Serum starvation and “ER shock” by the N-glycosylation blocker tunicamicin further shifted inactivation to negative voltages. The average V1/2 was −59±12 mV (n=12) in serum-free and −69±13 mV(n=32) in tunicamicin treated cells. These treatments altered inactivation only when γ1 was present and the effects were similar when β3 substituted for β2a. Mutations of γ1 that remove consensus N-glycosylation sites had only partial effect (V1/2=-60±18 mV, n=29) and did not reduce the cell-to-cell variability, indicating that N-glycosylation of γ1 was not its primary cause.Serum starvation and tunicamicin are known to produce cell-cycle arrest in the G0/G1 phase and, therefore, could act on γ1 indirectly by interfering with a cell-cycle dependent pathway. We characterized inactivation in cells expressing fluorescent probes visualizing cell-cycle activity. In support of our hypothesis, V1/2 was −55±16 mV, n=32, in G1 and −36±7 mV, n=20, in S/G2/M cells.Therefore, we propose that a novel cell-cycle dependent regulatory pathway controls voltage-dependent inactivation and functional availability of L-type calcium channels in the presence of γ1 subunit.The project was supported by MH079406 from NIMH.

KCNH2 Channel Activators Increase Ikr in Hl-1 Cardiomyocytes and May Prevent the Occurrence of Torsades De Pointes in Long QT Syndrome

Introduction: Several KCNH2 channel activators may provide a novel pharmacological approach for the treatment of long QT syndrome (LQTS). We therefore investigated the effects of the activators on IKr and action potential of cardiac myocytes.Methods: We characterized the actions of three KCNH2 channel activators, mallotoxin (MTX), PD-118057 (PD), and NS1643 (NS) on IKr of HL-1 cardiomyocytes, using the whole cell patch clamp technique. With a mathematical model of human ventricular myocytes, we further evaluated the impact of activator-induced changes in IKr kinetics on the action potential configuration in normal and LQTS.Results: The maximum tail currents of IKr were 23.4±3.5 nA/pF with 10 μM MTX (n=12), 22.1±2.7 nA/pF with 10 μM PD (n=13), and 23.3±2.7 nA/pF with 10 μM NS (n=16), which were significantly greater than 12.8±1.0 nA/pF in control (n=38). The half-maximal activation voltage was significantly shifted from −1.8±2.7 (n=38) to −13.0±2.3 (n=11), −8.3±2.1 (n=13), and −14.7±3.2 (n=14) mV by MTX, PD, and NS, respectively. Deactivation during the repolarization to −40 mV was significantly slowed by MTX, but not by PD or NS. The half-maximal inactivation voltage was significantly shifted from −6.6±2.2 (n=28) to −29.9±2.9 (n=15) mV by MTX, but not by PD, and NS. Simulation study showed that the activator-induced changes of IKr increased the amplitude of IKr during phase 2 of action potentials and consequently shortened the action potential duration by 19.7-23.6% in LQT1 and LQT3 models. A reduction of IKr in the LQT3 model evoked early afterdepolarization, which was abolished by the activator-induced enhancement of IKr.Conclusion: KCNH2 channel activators, mallotoxin, PD-118057, and NS1643 increases IKr through distinct kinetic mechanisms and can be utilized for potential therapy of LQTS and torsades de pointes.

Brivaracetam (ucb 34714) inhibits Na + current in rat cortical neurons in culture

Brivaracetam (ucb 34714; BRV), a new antiepileptic drug (AED) candidate, is a pyrrolidone derivative displaying a markedly higher affinity than levetiracetam (LEV; Keppra) to the synaptic vesicle protein SV2A, shown to be the brain-specific binding site of LEV. The higher affinity for SV2A correlates significant antiepileptic activity in animal epilepsy models in vitro and in vivo. Since many AEDs act upon inhibiting neuronal Na(+) currents, this study explored putative activity of BRV on the properties of these currents. Voltage-activated Na(+) currents were recorded by whole-cell patch-clamp on neuronal somas of rat neocortical neurons, grown in dissociated cell culture for up to 12 days. BRV, dissolved at the desired final concentration (between 0.2microM and 1mM) was applied by a multi-barrel pipette system near the soma of the recorded neuron. BRV produced a concentration-dependent inhibition of voltage-dependent Na(+) currents with IC(50) values of 41microM at the holding potential of -100mV, and of 6.5microM at the holding potential of -60mV. The voltage-dependence of activation and the kinetics of fast inactivation were not modified in the presence of BRV (30microM). Conversely, the recovery from fast inactivation was significantly slower and the voltage of half-maximal inactivation was shifted toward hyperpolarized value after BRV perfusion in a concentration-dependent manner. Furthermore, BRV (30microM) induced a significant use-dependent block at 50Hz stimulation frequency. These results indicate that BRV is able to modulate the voltage-activated Na(+) inflow in cortical neurons, which conceivably might contribute to the antiepileptic activity of this drug.

Mallotoxin Is a Novel HumanEther-a-go-go-Related Gene (hERG) Potassium Channel Activator

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.

Inhibition of cardiac HERG channels by grapefruit flavonoid naringenin: implications for the influence of dietary compounds on cardiac repolarisation

Flavonoids are naturally occurring food ingredients that have been associated with reduced cardiovascular mortality in epidemiological studies. In a previous study, we demonstrated for the first time that flavonoids are inhibitors of cardiac human ether-à-go-go-related gene (HERG) channels. Furthermore, we observed that grapefruit juice induced mild QTc prolongation in healthy subjects. HERG blockade by grapefruit flavonoid naringenin is most likely to be the mechanism underlying this effect. Therefore, the electrophysiological properties of HERG blockade by naringenin were analysed in detail. HERG potassium currents expressed in Xenopus oocytes were measured with a two-microelectrode voltage clamp. Naringenin blocked HERG potassium channels with an IC50 value of 102.6 microM in Xenopus oocytes. The onset of blockade was fast. The effect was completely reversible upon wash-out. Naringenin binding to HERG required aromatic residue F656 in the putative pore binding site. Channels were blocked in the open and inactivated states but not in the closed states. Naringenin did not affect HERG current activation. However, the half maximal inactivation voltage was shifted by 14.9 mV towards more negative potentials and current inactivation at negative potentials was accelerated. No frequency dependence of blockade was observed. Naringenin inhibits HERG channels with pharmacological characteristics similar to those of well-known HERG antagonists. From a clinical point of view, this effect could have both proarrhythmic and antiarrhythmic consequences. This may have important implications for phytotherapy and for dietary recommendations for cardiologic patients. Therefore, electrophysiological effects of flavonoids deserve further investigation.

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents.

Kv4.3 channels conduct transient outward K(+) currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K(+) channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K(+) currents.

Effects of derivatives of sulfur dioxide on transient outward potassium currents in acutely isolated hippocampal neurons

The effect of SO 2 derivatives, a common air pollutant and exists in vivo as an equilibrium between bisulfate and sulfite, on transient outward currents (TOC S) in hippocampal neurons were studied using the whole cell configuration of patch-clamp technique. TOC S that preliminary included a fast inactivating (A-current or I A ) and a slow inactivating (D-current or I D ) current, were isolated based on the kinetics and pharmacological properties in the presence of 50 mM TEA. The results showed that SO 2 derivatives reversibly increased the amplitudes of TOC S in a concentration dependent and voltage dependent. Half-increase dose on TOC S was 25 μM. In vivo, SO 2 derivatives shifted the steady-state inactivation curve of TOC S in the depolarizing direction but had little effect on the activation curve. Half-maximal inactivation voltage of TOC S was −69.6 ± 1.0 mV before and −56.8 ± 0.4 mV after application of 10 μM SO 2 derivatives. SO 2 derivatives increased the maximal conductance and delayed the inactivation process of TOC S. These results suggest that SO 2 derivatives would increase the excitability of hippocampal neurons.

Inhibition of fast sodium current in rabbit ventricular myocytes by protein tyrosine kinase inhibitors.

The present study investigated the effects of protein tyrosine kinase inhibitors on the fast sodium current ( I(Na)) in rabbit ventricular myocytes. Single rabbit ventricular myocytes were isolated enzymatically using Langendorff perfusion. I(Na) was recorded using the whole-cell patch-clamp technique at room temperature. The protein tyrosine kinase inhibitors genistein, AG957, ST638, and PP2 reversibly inhibited I(Na) in a concentration-dependent manner. At a test pulse potential of -30 mV, genistein (n=7) inhibited I(Na) by 37.7+/-3.2%, 53.4+/-2.5%, and 71.8+/-2.7% at concentrations of 15, 50, and 100 microM, respectively, without changing the voltage dependence of activation, while 100 microM AG957, 100 microM ST638, and 30 microM PP2 inhibited I(Na) by 38.7+/-2.4, 35.8+/-3.4, and 21.1+/-3.9%, respectively. Genistein (100 microM) and AG957 (100 microM) shifted the voltage for half-maximal inactivation of I(Na) from -76.7+/-2.0 mV (n=10) in control to -88.37+/-2.6 mV (n=6, P<0.05), and -82.9+/-1.7 (n=4, P<0.05), respectively, without changing the slope factor. Genistein and AG957 also significantly prolonged the time course of I(Na) recovery from inactivation. Daidzein and PP3, inactive analogs of genistein and PP2, respectively, did not inhibit I(Na) significantly. We conclude that protein tyrosine kinase signaling pathways may play an important role in regulation of I(Na) in cardiac myocytes.

Ketamine effects on human neuronal Na+ channels.

Similar doses of ketamine are employed in regional and general anaesthesia. In contrast to commonly used local anaesthetic agents, accidental systemic application of local anaesthetic doses of ketamine will not result in seizure, dysrhythmia or cardiovascular depression. However, there is some doubt about the quality of regional analgesia induced by ketamine. As human sodium channels constitute an important molecular target of local anaesthetics, the study was designed to establish concentration-dependent effects of ketamine on conductance, steady-state activation and steady-state inactivation of human neuronal sodium channels. This information--that has, so far, not been published--will help to characterize further local anaesthetic properties of ketamine. Whole-cell patch-clamp recordings were made of sodium channels natively expressed in human neuroblastoma SH-SY5Y cells. The sodium channels activated at a threshold of -60 mV and exhibited a maximal peak current at -10 mV. The voltage of half-maximal activation was -20 mV. The Na+ currents depended on the prepulse potential. The voltage of half-maximal inactivation was -80 mV. Ketamine inhibited the sodium conductance in a concentration-dependent manner (IC50 = 1140 micromol). A concentration-dependent hyperpolarizing shift of both steady-state activation and steady-state inactivation accounted for at most 5 mV. The effects of ketamine on these human ion channels occur at clinical concentrations. They are consistent with the local anaesthetic action of ketamine. Whether ketamine helps to decrease the incidence of severe side-effects during regional anaesthesia needs to be addressed in further clinical studies.