Modulation of voltage-dependent sodium channels by the delta-agonist SNC80 in acutely isolated rat hippocampal neurons

Following activation, voltage-gated Na+ currents (I(Na)) inactivate on two different time scales: fast inactivation takes place on a time scale of milliseconds, while slow inactivation takes place on a time scale of seconds to minutes. Both fast and slow inactivation processes govern availability of Na+ channels. In this study, the effects of the delta-opioid receptor agonist SNC80 on slow and fast inactivation of I(Na) in rat hippocampal granule cells were analyzed in detail. Following application of SNC80, a block of the peak Na+ current amplitude (EC50: 50.6 microM, Hill coefficient: 0.518) was observed. Intriguingly, SNC80 (50 microM) also caused a selective effect on slow but not fast inactivation processes, with a notable increase in the fraction of Na+ channels undergoing slow inactivation during prolonged depolarization. In addition, recovery from slow inactivation was considerably slowed. At the same time, fast recovery processes were unaffected. The effects of SNC80 were not mimicked by the peptide delta-receptor agonist DPDPE (10 microM), and were not inhibited by the opioid receptor antagonists naloxone (50-300 microM) or naltrindole (10 and 100 microM), indicating an opioid receptor independent modulation of Na+ channels. These data suggest that SNC80 not only affects delta-opioid receptors, but also voltage-gated Na+ channels. SNC80 is to our knowledge hitherto the only substance that selectively influences slow but not fast inactivation processes and could provide an important tool in unraveling the mechanism underlying these distinct biophysical processes.     

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.     

NA+- and K+-channels as molecular targets of the alkaloid ajmaline in skeletal muscle fibres

BACKGROUND AND PURPOSE: Ajmaline is a widely used antiarrhythmic drug. Its action on voltage-gated ion channels in skeletal muscle is not well documented and we have here elucidated its effects on Na(+) and K(+) channels. EXPERIMENTAL APPROACH: Sodium (I(Na)) and potassium (I(K)) currents in amphibian skeletal muscle fibres were recorded using 'loose-patch' and two-microelectrode voltage clamp techniques (2-MVC). Action potentials were generated using current clamp. KEY RESULTS: Under 'loose patch' clamp conditions, the IC(50) for I(Na) was 23.2 microM with Hill-coefficient h=1.21. For I(K), IC(50) was 9.2 microM, h=0.87. Clinically relevant ajmaline concentrations (1-3 microM) reduced peak I(Na) by approximately 5% but outward I(K) values were reduced by approximately 20%. Na(+) channel steady-state activation and fast inactivation were concentration-dependently shifted towards hyperpolarized potentials ( approximately 10 mV at 25 microM). Inactivation curves were markedly flattened by ajmaline. Peak-I(K) under maintained depolarisation was reduced to approximately 30% of control values by 100 microM ajmaline. I(K) activation time constants were increased at least two-fold. Lower concentrations (10 or 25 microM) reduced steady-state-I(K) slightly but peak-I(K) significantly. Action potential generation threshold was increased by 10 microM ajmaline and repolarisation prolonged. CONCLUSIONS AND IMPLICATIONS: Ajmaline acts differentially on Na(+) and K(+) channels in skeletal muscle. This suggests at least multiple sites of action including the S4 subunit. Our data may provide a first insight into specific mechanisms of ajmaline-ion channel interaction in tissues other than cardiac muscle and could suggest possible side-effects that need to be further evaluated.     

V102862 (Co 102862): a potent, broad-spectrum state-dependent blocker of mammalian voltage-gated sodium channels

1. 4-(4-Fluorophenoxy)benzaldehyde semicarbazone (V102862) was initially described as an orally active anticonvulsant with robust activity in a variety of rodent models of epilepsy. The mechanism of action was not known. We used whole-cell patch-clamp techniques to study the effects of V102862 on native and recombinant mammalian voltage-gated Na+ channels. 2. V102862 blocked Na+ currents (I(Na)) in acutely dissociated cultured rat hippocampal neurons. Potency increased with membrane depolarization, suggesting a state-dependent mechanism of inhibition. There was no significant effect on the voltage dependence of activation of I(Na). 3. The dissociation constant for the inactivated state (K(I)) was approximately 0.6 microM, whereas the dissociation constant for the resting state (K(R)) was >15 microM. 4. The binding to inactivated channels was slow, requiring a few seconds to reach steady state at -80 mV. 5. The mechanism of inhibition was characterized in more detail using human embryonic kidney-293 cells stably expressing rat brain type IIA Na+ (rNa(v)1.2) channels, a major Na+ channel alpha subunit in rat hippocampal neurons. Similar to hippocampal neurons, V102862 was a potent state-dependent blocker of rNa(v)1.2 channels with a K(I) of approximately 0.4 microM and K(R) approximately 30 microM. V102862 binding to inactivated channels was relatively slow (k(+) approximately = 1.7 microM(-1) s(-1)). V102862 shifted the steady-state availability curve in the hyperpolarizing direction and significantly retarded recovery of Na+ channels from inactivation. 6. These results suggest that inhibition of voltage-gated Na+ channels is a major mechanism underlying the anticonvulsant properties of V102862. Moreover, understanding the biophysics of the interaction may prove to be useful in designing a new generation of potent Na+ channel blocker therapeutics.     

Cardiac electrophysiologic and antiarrhythmic actions of a pavine alkaloid derivative,O-methyl-neocaryachine,in rat heart

1. O-methyl-neocaryachine (OMNC) suppressed the ischaemia/reperfusion-induced ventricular arrhythmias in Langendorff-perfused rat hearts (EC50=4.3 microM). Its electrophysiological effects on cardiac myocytes and the conduction system in isolated hearts as well as the electromechanical effects on the papillary muscles were examined. 2. In rat papillary muscles, OMNC prolonged the action potential duration (APD) and decreased the maximal rate of depolarization (V(max)). As compared to quinidine, OMNC exerted less effects on both the V(max) and APD but a positive inotropic effect. 3. In the voltage clamp study, OMNC decreased Na+ current (I(Na)) (IC50=0.9 microM) with a negative-shift of the voltage-dependent inactivation and a slowed rate of recovery from inactivation. The voltage dependence of I(Na) activation was, however, unaffected. With repetitive depolarizations, OMNC blocked I(Na) frequency-dependently. OMNC blocked I(Ca) with an IC(50) of 6.6 microM and a maximum inhibition of 40.7%. 4. OMNC inhibited the transient outward K+ current (I(to)) (IC50=9.5 microM) with an acceleration of its rate of inactivation and a slowed rate of recovery from inactivation. However, it produced little change in the steady-state inactivation curve. The steady-state outward K+ current (I(SS)) was inhibited with an IC50 of 8.7 microM. The inward rectifier K+ current (I(K1)) was also reduced by OMNC. 5. In the perfused heart model, OMNC (3 to 30 microM) prolonged the ventricular repolarization time, the spontaneous cycle length and the atrial and ventricular refractory period. The conduction through the AV node and His-Purkinje system, as well as the AV nodal refractory period and Wenckebach cycle length were also prolonged (30 microM). 6. In conclusion, OMNC blocks Na+, I(to) and I(SS) channels and in similar concentrations partly blocks Ca2+ channels. These effects lead to a modification of the electromechanical function and may likely contribute to the termination of ventricular arrhythmias. These results provide an opportunity to develop an effective antiarrhythmic agent with modest positive inotropy as well as low proarrhythmic potential.     

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells.

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.     

Inhibition by clonidine of the carbachol-induced tension development and nonselective cationic current in guinea pig ileal myocytes.

Effects of clonidine, an imidazoline derivative as well as alpha2-adrenoceptor agonist, on carbachol (CCh)-evoked contraction in guinea pig ileal smooth muscle were studied using isometric tension recording. To investigate the cellular mechanisms of the inhibitory effect of clonidine, its effects on CCh-evoked nonselective cationic current (I(CCh)), voltage-dependent Ca2+ current (I(Ca)) and voltage-dependent K+ current (I(K)) was also studied using patch-clamp recording techniques in single ileal cells. Clonidine inhibited the contraction evoked by CCh (1 microM) in a concentration-dependent manner with an IC50 valve of 61.7 +/- 2.5 microM. High K+ (40 mM)-evoked contraction was only slightly inhibited even when clonidine was used at 300 microM. Externally applied clonidine inhibited I(CCh) dose-dependently with an IC50 of 42.0 +/- 2.6 microM. When applied internally via patch pipettes, clonidine was without effect. An I(CCh)-like current induced by GTPgammaS was also inhibited by bath application of clonidine. None of KU14R and BU224, both imidazoline receptor blockers, and yohimbine, an alpha2-adrenergic blocker, significantly affects the inhibitory effect of clonidine on I(CCh). Clonidine (300 microM) only slightly decreased membrane currents flowing through voltage-gated Ca2+ channels or K+ channels. These data indicate that clonidine relaxes smooth muscle contraction produced by muscarinic receptor activation and suggest that the effect of clonidine seems due mainly to inhibition of I(CCh) via acting directly on the involved cationic channel.     

Isoflurane and propofol inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve terminals

Mounting electrophysiological evidence indicates that certain general anesthetics, volatile anesthetics in particular, depress excitatory synaptic transmission by presynaptic mechanisms. We studied the effects of representative general anesthetics on voltage-gated Na+ currents (INa) in nerve terminals isolated from rat neurohypophysis using patch-clamp electrophysiological analysis. Both isoflurane and propofol inhibited INa in a dose-dependent and reversible manner. At holding potentials of -70 or -90 mV, isoflurane inhibited peak INa with IC50 values of 0.45 and 0.56 mM, and propofol inhibited peak INa with IC50 values of 4.1 and 6.0 microM, respectively. Isoflurane (0.8 mM) did not significantly alter the V1/2 of activation; propofol caused a small positive shift. Isoflurane (0.8 mM) or propofol (5 microM) produced a negative shift in the voltage dependence of inactivation. Recovery of INa from inactivation was slower from a holding potential of -70 mV than from -90 mV; isoflurane and propofol further delayed recovery from inactivation. In conclusion, the volatile anesthetic isoflurane and the intravenous anesthetic propofol inhibit voltage-gated Na+ currents in isolated neurohypophysial nerve terminals in a concentration- and voltage-dependent manner. Marked effects on the voltage dependence and kinetics of inactivation and minimal effects on activation support preferential anesthetic interactions with the fast inactivated state of the Na+ channel. These results are consistent with direct inhibition of oxytocin and vasopressin release from the neurohypophysis by isoflurane and propofol. Inhibition of voltage-gated Na+ channels may contribute to the presynaptic effects of general anesthetics on nerve terminal excitability and neurotransmitter release.     

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.     

N-salicyloyltryptamine, a new anticonvulsant drug, acts on voltage-dependent Na+, Ca2+, and K+ ion channels

1. The aim of this work was to study the effects of N-salicyloyltryptamine (STP), a novel anticonvulsant agent, on voltage-gated ion channels in GH3 cells. 2. In this study, we show that STP at 17 microM inhibited up to 59.2+/-10.4% of the Ito and 73.1+/-8.56% of the IKD K+ currents in GH3 cells. Moreover, the inhibitory activity of the drug STP on K+ currents was dose-dependent (IC50=34.6+/-8.14 microM for Ito) and partially reversible after washing off. 3. Repeated stimulation at 1 Hz (STP at 17 microM) led to the total disappearance of Ito current, and an enhancement of IKD. 4. In the cell-attached configuration, application of STP to the bath increased the open probability of large-conductance Ca2+-activated K+ channels. 5. STP at 17 microM inhibited the L-type Ca2+ current by 54.9+/-7.50% without any significant changes in the voltage dependence. 6. STP at 170 microM inhibited the TTX-sensitive Na+ current by 22.1+/-2.41%. At a lower concentration (17 microM), no effect on INa was observed. 7. The pharmacological profile described here might contribute to the neuroprotective effect exerted by this compound in experimental 'in vivo' models.     

Inhibition of voltage-gated calcium channels by fluoxetine in rat hippocampal pyramidal cells

Fluoxetine, an antidepressant which is used world-wide, is a prominent member of the class of selective serotonin re-uptake inhibitors. Recently, inhibition of voltage-gated Na(+) and K(+) channels by fluoxetine has also been reported. We examined the effect of fluoxetine on voltage-gated calcium channels using the patch-clamp technique in the whole-cell configuration.In hippocampal pyramidal cells, fluoxetine inhibited the low-voltage-activated (T-type) calcium current with an IC(50) of 6.8 microM. Fluoxetine decreased the high-voltage-activated (HVA) calcium current with an IC(50) between 1 and 2 microM. Nifedipine and omega-conotoxin GVIA inhibited the HVA current by 24% and 43%, respectively. Fluoxetine (3 microM), applied in addition to nifedipine or omega-conotoxin, further reduced the current. When fluoxetine (3 microM) was applied first neither nifedipine nor omega-conotoxin attenuated the remaining component of the HVA current. This observation indicates that fluoxetine inhibits both L- and N-type currents.In addition, fluoxetine inhibited the HVA calcium current in carotid body type I chemoreceptor cells and pyramidal neurons prepared from prefrontal cortex. In hippocampal pyramidal cells high K(+)-induced seizure-like activity was inhibited by 1 microM fluoxetine; the mean burst duration was shortened by an average of 44%.These results provide evidence for inhibition of T-, N- and L-type voltage-gated calcium channels by fluoxetine at therapeutically relevant concentrations.     

Properties of voltage-gated Na+ channels in the human rhabdomyosarcoma cell-line SJ-RH30: conventional and automated patch clamp analysis.

Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current-voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.     

Verapamil inhibits proliferation of LNCaP human prostate cancer cells influencing K+ channel gating

The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K+ currents (I(K)) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type) inactivation. With 2 mM [Mg(2+)](o), I(K) activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC(50) = 11 microM) rapidly (12 s) inhibited I(K) in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 microM) did not influence whole-cell I(K), although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 microM) had no influence on I(K). Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I(K) were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 +/- 0.5 to 3.2 +/- 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 microM) reduced an open-channel probability (from 0.45 +/- 0.1 in control to 0.1 +/- 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.     

Inhibition of delayed rectifier K+ currents by copper in acutely dissociated rat hippocampal CA1 neurons

A growing number of research results demonstrate that copper is an important trace element to life. In this study, whole-cell recording made from acutely dissociated rat hippocampal CA1 neurons was employed to investigate the actions of copper (Cu(2+)) on the delayed rectifier K(+) currents (I(K)). External application of various concentrations of Cu(2+) (1-1000microM) reduced the amplitude of I(K) in a dose-dependent manner with an IC(50) value of 100microM and a Hill coefficient of 0.4. 300microM of Cu(2+) depolarized the I(K) activation curves by 12.5mV and hyperpolarized the I(K) state-inactivation curves by 17.4mV, respectively. At this concentration, Cu(2+) also significantly increased the value of the fast decay time constant (tau(1)), but had no effect on the I(K) recovery from inactivation. These results suggest that relevant concentrations of copper at physiological and pathological level can influence the neuronal excitability of rat hippocampal CA1 neurons by voltage-gated delayed rectifier K(+) channels, and such actions are likely involved in the pathophysiology of Cu-related Wilson's disease.     

The mechanism of inhibitory actions of S-petasin,a sesquiterpene of Petasites formosanus,on L-type calcium current in NG108-15 neuronal cells

The effects of S-petasin, a sesquiterpene isolated from Petasites formosanus Kitamura, on ion currents in a mouse neuroblastoma and a rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. S-Petasin (1 - 300 microM) caused a decrease in the amplitude of L-type Ca2+ current (I(Ca,L)) in a concentration-dependent manner, however, it did not change the overall shape of the current-voltage relationship of I(Ca,L). The IC50 value for S-petasin-induced inhibition of I(Ca,L) was 11 microM. S-Petasin (10 microM) shifted the steady-state inactivation of I(Ca,L) to a more negative membrane potential by approximately -10 mV. S-petasin could prolong the recovery of I(Ca,L) inactivation. The inhibitory effect of S-petasin on I(Ca,L) was found to exhibit tonic and use-dependent characteristics. S-Petasin could inhibit I(Ca,L) evoked by action potential waveforms effectively. S-Petasin also suppressed low voltage-activated I(Ca,L) in NG108-15 cells. S-Petasin at a concentration of 100 microM had little effect on voltage-dependent Na+ current; however, it did produce an inhibitory effect on delayed rectifier K+ current in a time-dependent manner. These results demonstrate that S-petasin can interact directly with L-type Ca2+ channels in NG108-15 cells. These effects could contribute to the regulation of neuronal activity if similar results were found in neurons in vivo.     

Actions of the novel neuroprotective agent, lifarizine (RS-87476), on voltage-dependent sodium currents in the neuroblastoma cell line, N1E-115.

1. The actions of the neuroprotective agent, lifarizine (RS-87476-190), on voltage-dependent Na+ currents have been examined in the neuroblastoma cell line, N1E-115, using the whole-cell variant of the patch clamp technique. 2. At a holding potential of -80 mV, lifarizine reduced the peak Na+ current evoked by a 10 ms depolarizing step with an IC50 of 1.3 microM. At holding potentials of -100 and -60 mV the IC50 concentrations of lifarizine were 7.3 microM and 0.3 microM, respectively. 3. At a holding potential of -100 mV, most channels were in the resting state and the IC50 value for inhibition of Na+ current should correspond to the dissociation constant of lifarizine for resting channels (KR). KR was therefore estimated to be 7.3 microM. 4. In the absence of lifarizine, recovery from inactivation following a 20 s depolarization from -100 mV to 0 mV was complete within 2 s. However, in the presence of 3 microM lifarizine recovery took place in a biexponential fashion with time constants of 7 s and 79 s. 5. Lifarizine (1 microM) had no effect on steady-state inactivation curves when conditioning pre-pulses of 1 s duration were used. However, when pre-pulse durations of 1 min were used the curves were shifted to the left by lifarizine by about 10 mV. Analysis of the shifts induced by a range of lifarizine concentrations revealed that the apparent affinity of lifarizine for the inactivated state of the channel (K1) was 0.19 microM. 6. Lifarizine (1 microM) had no effect on chloramine-T-modified Na+ currents, suggesting no significant open channel interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by fluoxetine of voltage-activated ion channels in rat PC12 cells

The effects of fluoxetine (Prozac) on voltage-activated K+, Ca2+ and Na+ channels were examined using the whole-cell configuration of the patch clamp technique in rat pheochromocytoma (PC12) cells. When applied to the external bath solution, fluoxetine (1, 10, 100 microM) decreased the peak amplitude of K+ currents. The K+ current inhibition by fluoxetine (10 microM) was voltage-independent and the fraction of current inhibition was 39.7-51.3% at all voltages tested (0 to +50 mV). Neither the activation and inactivation curves nor the reversal potential for K+ currents was significantly changed by fluoxetine. The inhibition by fluoxetine of K+ currents was use- and concentration-dependent with an IC50 of 16.0 microM. The inhibition was partially reversible upon washout of fluoxetine. The action of fluoxetine was independent of the protein kinases, because the protein kinase C or A inhibitors (H-7, staurosporine, Rp-cAMPS) did not prevent the inhibition by fluoxetine. Intracellular infusion with GDPbetaS or pretreatment with pertussis toxin did not block the inhibitory effects of fluoxetine. The inhibitory action of fluoxetine was not specific to K+ currents because it also inhibited both Ca2+ (IC50 = 13.4 microM) and Na+ (IC50 = 25.6 microM) currents in a concentration-dependent manner. Our data indicate that when applied to the external side of cells, fluoxetine inhibited voltage-activated K+, Ca2+ and Na+ currents in PC12 cells and its action on K+ currents does not appear to be mediated through protein kinases or G proteins.     

Assembly with the Kvbeta1.3 subunit modulates drug block of hKv1.5 channels

The assembly of voltage-gated potassium (Kv) channels with beta subunits modifies the electrophysiological characteristics of the alpha subunits. Kvbeta1.3 subunits shift the midpoint of the activation curve toward more negative voltages and slow the deactivation process. In addition, the Kvbeta1.3 subunit converts hKv1.5 from a delayed rectifier with a modest degree of slow inactivation to a channel with both fast and slow components of inactivation. In the present study, we have analyzed the effects of bupivacaine and a permanently charged analog [R(+)-N-methyl-bupivacaine (RB(+)1C)] on Kvalpha1.5 and Kvalpha1.5+Kvbeta1.3 channels expressed in human embryonic kidney 293 cells using the whole-cell configuration of the patch-clamp technique. Block induced by RB(+)1C binding to its external receptor site was not modified by the presence of this beta subunit. However, hKvalpha1.5+Kvbeta1.3 channels were ~4-fold less sensitive to bupivacaine than hKv1.5 channels in the absence of beta subunits (IC(50) = 47.5 +/- 5.1 versus 13.1 +/- 0.8 microM, respectively, p < 0.01). Quinidine was also less potent to block Kvalpha1.5+Kvbeta1.3 channels than Kvalpha1.5 channels (IC(50) = 49.6 microM versus 6.2 microM, respectively). These results suggest that the Kvbeta1.3 subunit does not modify the affinity of the charged bupivacaine for its external receptor site but markedly reduces the affinity of bupivacaine and quinidine for their internal receptor site in hKv1.5 channels.     

Inhibition of voltage-gated potassium currents by gambierol in mouse taste cells.

Ciguatera is a food poisoning caused by toxins of Gambierdiscus toxicus, a marine dinoflagellate. The neurological features of this intoxication include sensory abnormalities, such as paraesthesia, heightened nociperception, and also taste alterations. Here, we have evaluated the effect of gambierol, one of the possible ciguatera toxins, on the voltage-gated ion currents in taste cells. Taste cells are excitable cells endowed with voltage-gated Na+, K+, and Cl- currents (I(Na), I(K), and I(Cl), respectively). By applying the patch-clamp technique to single cells in isolated taste buds obtained from the mouse vallate papilla, we have recorded such currents and determined the effect of bath-applied gambierol. We found that this toxin markedly inhibited I(K) in the nanomolar range (IC50 of 1.8 nM), whereas it showed no significant effect on I(Na) or I(Cl) even at high concentration (1 microM). The block of I(K) was irreversible even after a 50-min wash. In addition to affecting the current amplitude, we found that gambierol significantly altered both the activation and inactivation processes of I(K). In conclusion, unlike other toxins involved in ciguatera, such as ciguatoxins, which affect the functioning of voltage-gated sodium channels, the preferred molecular target of gambierol is the voltage-gated potassium channel, at least in taste cells. Voltage-gated potassium currents play an important role in the generation of the firing pattern during chemotransduction. Thus, gambierol may alter action potential discharge in taste cells and this could be associated with the taste alterations reported in the clinical literature.     

Donepezil attenuates excitotoxic damage induced by membrane depolarization of cortical neurons exposed to veratridine

Long-lasting membrane depolarization in cerebral ischemia causes neurotoxicity via increases of intracellular sodium concentration ([Na+]i) and calcium concentration ([Ca2+]i). Donepezil has been shown to exert neuroprotective effects in an oxygen-glucose deprivation model. In the present study, we examined the effect of donepezil on depolarization-induced neuronal cell injury resulting from prolonged opening of Na+ channels with veratridine in rat primary-cultured cortical neurons. Veratridine (10 microM)-induced neuronal cell damage was completely prevented by 0.1 microM tetrodotoxin. Pretreatment with donepezil (0.1-10 microM) for 1 day significantly decreased cell death in a concentration-dependent manner, and a potent NMDA receptor antagonist, dizocilpine (MK801), showed a neuroprotective effect at the concentration of 10 microM. The neuroprotective effect of donepezil was not affected by nicotinic or muscarinic acetylcholine receptor antagonists. We further characterized the neuroprotective properties of donepezil by measuring the effect on [Na+]i and [Ca2+]i in cells stimulated with veratridine. At 0.1-10 microM, donepezil significantly and concentration-dependently reduced the veratridine-induced increase of [Ca2+]i, whereas MK801 had no effect. At 10 microM, donepezil significantly decreased the veratridine-induced increase of [Na+]i. We also measured the effect on veratridine-induced release of the excitatory amino acids, glutamate and glycine. While donepezil decreased the release of glutamate and glycine, MK801 did not. In conclusion, our results indicate that donepezil has neuroprotective activity against depolarization-induced toxicity in rat cortical neurons via inhibition of the rapid influx of sodium and calcium ions, and via decrease of glutamate and glycine release, and also that this depolarization-induced toxicity is mediated by glutamate receptor activation.