Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Comparison of the electrophysiological effect of amiodarone, lidocaine and quinidine on rat ventricular cells.

The effects of 20 microM each of amiodarone, lidocaine and quinidine on action potential and membrane currents were studied in rat ventricular cells. At a stimulation frequency of 0.1 Hz, quinidine prolonged the action potential duration (APD50) from 120 +/- 26 to 660 +/- 8 msec and increased the time to peak (Tp) amplitude from 7 +/- 1 msec to 32 +/- 6 msec. Lidocaine shortened APD50 from 123 +/- 15 to 83 +/- 6 msec without altering Tp. Amiodarone changed neither APD50 nor Tp. Voltage clamp study revealed that quinidine inhibited sodium inward current (INa) even when this current was elicited by depolarizing pulses at 0.1 Hz from a holding potential of -90 mV. For amiodarone and lidocaine, the inhibition was observed when INa was elicited from a holding potential of -70 mV. A frequency-dependent inhibition of INa by amiodarone and lidocaine was observed at frequencies higher than 1 Hz. Quinidine showed this inhibition even at 1 Hz. In correlation with the stronger frequency dependent inhibition of INa, a greater delay of the recovery and increase of the non-recovery fraction of INa was induced by quinidine. For lidocaine and amiodarone, only the recovery time constant was delayed. In cells treated with sea anemone toxin (ATX, 0.2 microM), APD50 was prolonged to 4-5 sec in 5 min. Quinidine, but not amiodarone, completely reversed the effect of ATX. Quinidine showed use-dependent inhibition of INa in these ATX-treated cells. Amiodarone, however, did not show this inhibition. It is likely that amiodarone suppresses INa by delaying the recovery of INa instead of blocking the open-state Na(+)-channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model of the electrophysiological properties of nucleus reticularis thalami neurons.

A model of the electrophysiological properties of rodent nucleus reticularis thalami (NRT) neurons of the dorsal lateral thalamus was developed using Hodgkin-Huxley style equations. The model incorporated voltage-dependent rate constants and kinetics obtained from recent voltage-clamp experiments in vitro. The intrinsic electroresponsivity of the model cell was found to be similar to several empirical observations. Three distinct modes of oscillatory activity were identified: 1) a pattern of slow rhythmic burst firing (0.5-7 Hz) usually associated with membrane potentials negative to approximately -70 mV which resulted from the interplay of ITs and IK(Ca); 2) at membrane potentials from approximately -69 to -62 mV, rhythmic burst firing in the spindle frequency range (7-12 Hz) developed and was immediately followed by a tonic tail of single spike firing after several bursts. The initial bursting rhythm resulted from the interaction of ITs and IK(Ca), with a slow after-depolarization due to ICAN which mediated the later tonic firing; 3) with further depolarization of the membrane potential positive to approximately -61 mV, sustained tonic firing appeared in the 10-200-Hz frequency range depending on the amplitude of the injected current. The frequency of this firing was also dependent on the maximum conductance of the leak current, IK(leak), and an interaction between the fast currents involved in generating action potentials, INa(fast) and IK(DR), and the persistent Na+ current, INa(P). Transitions between different firing modes were identified and studied parametrically.     

Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl- aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX- B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX- B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.     

Autoregulation of apical sodium entry in the colon of the frog (Rana esculenta)

1. Na transport (INa) in the K-depolarized colon of the frog was investigated by electro-physiological current-voltage analysis. 2. INa and the intracellular Na activity [(Na)c] increased with increasing mucosal Na concentration ([Na]m), whereas the apical Na-permeability (PNam) and the transepithelial resistance (RT) decreased. 3. The results are consistent with a Na self-inhibition mechanism; however, a feedback inhibition of INa by intracellular Na must also be considered.     

二乙基二硫代氨基甲酸钠对二氧化硫衍生物引起的心肌细胞钠电流增大的增强效应

超氧化物歧化酶(superoxide dismutase,SOD)是生物体内专一的过氧自由基(superoxide anions,O2.-)清除剂,而二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDC)则是公认的Cu,Zn-SOD的抑制剂。采用全膜片钳技术研究了DDC对二氧化硫(sulfur dioxide,SO2)衍生物引起的大鼠心肌细胞钠电流增大效应的作用,以期更进一步揭示SO2的毒性机理。结果表明:SO2衍生物对SOD活性无显著影响,SO2衍生物存在时,DDC仍可以显著降低SOD的活性。DDC(10￣100 mmol/L)剂量依赖性地增大钠电流(INa),半数效应浓度为(19.85±0.95)mmol/L。将20 mmol/L的DDC与10μmol/L的SO2衍生物同时作用于心肌细胞,INa仍表现为电压依赖性的增大,并使INa的电压依赖性激活曲线显著地向负电压方向移动,稳态失活曲线向正电压方向移动,差异极其显著。这表明DDC增强了SO2衍生物对心肌细胞钠电流的增大效应,提示SO2衍生物引起的大鼠心肌细胞毒性主要是通过自由基,特别是O2.-氧化损伤实现的。     

Changes in the sodium current under the action of etmozin and lidocaine inside and outside the membrane of single rat myocardial cells

The voltage clamp experiments were carried out on single internally perfused rat myocardial cells. The effect of ethmozine (8 x 10(-5) g/ml) and lidocain (8 x 10(-6) g/ml) on the fast maximum inward sodium current (INa) was studied. The drugs were tested inside and outside the cell. INa was inhibited insignificantly when ethmozine was added inside the cell. After 5 min of ethmozine action outside the cell INa dropped on the average to 43 +/- 6% of its initial value. Under these conditions the reactivation constrant of INa did not change significantly. Lidocain depressed INa both when added outside and inside the cell. However, when lidocain was added outside the cell a longer period was needed to depress INa. Comparison of lidocain and ethmozine action outside and inside the myocardial cell has shown that the sites of action of these antiarrhythmic drugs on the cellular membrane are different.     

SUN 1165: a new antiarrhythmic Na current blocker in ventricular myocytes of guinea-pig

1. We compared the effect of a new antiarrhythmic compound, SUN 1165, on Na and Ca channels in papillary muscles and enzymatically dispersed single ventricular cells of guinea-pig. Action potential and contractile force in papillary muscle were measured by the conventional microelectrode technique and a strain gauge. The membrane currents were measured in internally perfused and voltage clamped cells by a single suction pipette technique. 2. In papillary muscles, SUN 1165 depressed the maximum rate of rise of action potential (Vmax) in a concentration dependent manner (IC30 = 1.7 X 10(-5) M) more markedly (about six times) than the contractile force. 3. In single ventricular cells, the Na current (INa) was reduced by the drug in a concentration dependent manner (IC30 = 9.1 X 10(-6) M). 4. It showed frequency-dependent block and the steady-state inactivation curve was shifted to more negative potentials. 5. The recovery of INa from inactivation was prolonged by SUN 1165. 6. The Ca current (ICa) was also blocked by the drug in a concentration dependent manner but much less than INa (IC30 = 5.5 X 10(-5) M). 7. These results suggested that SUN 1165 causes a selective inhibition of Na channels in guinea-pig ventricular cells at the antiarrhythmic concentrations.     

Temperature Dependence of Bistability in Squid Giant Axons with Alkaline Intracellular pH

Raising the intracellular pH (pHi) above 7.7 in intracellularly perfused squid giant axons causes spontaneous firing of action potentials. The firing frequency ranged from 20 Hz at 0 degrees C to 200 Hz at 23 degrees C. Above 23 degrees C, the axons were quiescent. They were bistable for 13 相似文献   

The effect of metacaine on the slow variation of peak sodium currents in potential clamped Ranvier nodes following changes in holding potential

The effect of changes in the holding potential on peak sodium currents in isolated myelinated nerve fibres (peak INa) was investigated with the conventional sodium inactivation being kept at h infinity = 1. In Ringer solution no stationary values of peak INa could be obtained over the potential range tested. Near the normal resting potential, ER, peak INa changed with time clearly even after 10 min. Therefore, the individual values of peak INa as normalized by peak INa at ER and corrected for the unevitable run-down of peak INa could not serve as measure for stationary values of any membrane parameter. Under metacaine (1 mmol/l) peak INa changed comparably faster and proved to be less potential dependent as compared to peak INa of the untreated fibre. The effects observed are not necessarily governed by a specific process located inside the nodal membrane.     

Effect of diethylamine analog of ethmozine on parameters of sodium current in isolated rat cardiomyocytes

Voltage clamp experiments were made on ezymically isolated and internally perfused rat cardiac cells. The effect of a diethylamine analog of ethmozine (DAAE) on sodium current (INa) was tested when the drug was applied inside or outside the cell. It was found that the effect of DAAE (8 X 10(-6) g/ml) on INa was asymmetrical: after DAAE addition outside the cell, the amplitude of INa was effectively suppressed. Thus, 5 minutes after DAAE action the maximal value of INa in a voltage-current relationship was 20% of the control value without significant changes in the kinetics of INa. When the DAAE was added inside the cell preferentially, the inactivation time constant was increased without significant changes in the amplitude of the maximal INa. The same results were obtained with pronase (1 mg/ml) added inside the cell. It was supposed that as compared to ethmozine, the DAAE possesses a supplementary binding site on the cardiac cell membrane possibly linked to the structures responsible for inactivation processes.     

Kinetic analysis of sodium channel block by internal methylene blue in pronased crayfish giant axons

The cationic dye methylene blue (MB+) blocks INa in a voltage and time-dependent manner and exhibits no frequency dependent block at 1 Hz when internally perfused in normal or pronase-treated crayfish axons. Peak INa decreases with increasing MB+ concentrations in the range 50 microM to 5 mM, but the blocking time constant approaches an asymptote at concentrations above 500 microM. IgON is not noticeably affected by internal MB+ at concentrations of 500 microM or below, in the absence of external tetrodotoxin (TTX). However, 5 mM MB+ produces a visible suppression of IgON that is reversible following washout. A pseudo-first-order analysis of MB+ blocking kinetics suggests a drug binding site deep in the transmembrane voltage field (dz = 0.85, KD = 11 microM at 0 mV). The voltage sensitivity of the individual rate constants is highly asymmetric, suggesting that the major energy barrier for MB+ is very close to the axoplasmic margin of the voltage field. Reversing the Na+ gradient and direction of INa has little effect on the kinetics of MB+ block. The kinetic properties of state-dependent vs. state-independent blocking schemes are investigated and compared with our observations of MB+ block. Analysis of hooked sodium tail currents following depolarization to various test potentials demonstrates quantitatively that MB+ binds in a state-dependent manner to open sodium channels. The appropriateness of first-order kinetic analysis of drug block is then considered in light of these observations.     

Effects of membrane potential on mechanical activation in skeletal muscle

The effect of subthreshold depolarization on mechanical threshold was investigated in tetrodotoxin-poisoned mammalian and amphibian skeletal muscle fibers using a two-microelectrode voltage-clamp technique. Mechanical threshold was determined with a 2-ms test pulse. The immediate effect of depolarization was inhibition of the mechanical system. The consequent increase in the test pulse threshold was linearly related to the size of the depolarization and there was, on the average, a 10% increase in threshold for a 10-mV depolarization in mammalian fibers. The duration of the inhibitory period was also related to the size of the depolarization. Inhibition was interrupted by the onset of activation (seen as a reduction in the test pulse threshold), and in rat soleus fibers this occurred within 100 ms with a 20-mV depolarization, inhibition decayed within 10 ms. The decay of activation after brief conditioning pulses was initially rapid (on the average, the test pulse threshold recovered to 80% of its control value within 1 ms) and then slow (full recovery took 100-500 ms). After long conditioning pulses, activation often decayed into a period of inhibition. When depolarization (of 20 mV or more) was maintained for several seconds, the fibers became inactivated. Rat extensor digitorum longus and sternomastoid fibers were strongly inactivated by depolarization to -40 mV and the test pulse to +40 mV did not cause contraction.     

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells.

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.     

Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres.

The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.