Ion currents through batrachotoxin-modified sodium channels of node of Ranvier membranes at high positive and negative potentials

Ionic currents through batrachotoxin-modified sodium channels in frog nerve fibres were measured over a wide range of membrane potentials. At potentials above +80 mV currents decay in time and their steady-state level decreased as potentials increased. "Instantaneous" current measurements have shown that this phenomenon was due to the decrease in net channel conductance. Scorpion toxin affected current kinetics only slightly at these potentials, which suggested that these decays were not caused by usual inactivation process. Externally applied procaine induced slow (tens of ms) potential-dependent block of batrachotoxin-modified channels at large positive potentials. At large negative potentials (above -100 mV) "instantaneus" currents decreased due to fast voltage-dependent block of the channels by calcium ions.     

Selectivity and sensitivity to blocking by hydrogen ions of batrachotoxin-modified sodium channels in nerve fiber membranes

Currents through normal and batrachotoxin-modified sodium channels in frog nerve were measured under voltage clamp conditions. Measured reversal potentials and the Goldman equation were used to calculate relative permeabilities. The permeability ratios were: PNa: PNH4: PK = 1: 0.47: 0.19. Hydrogen-to-sodium permeability ratio was estimated from reversal potential measurements in Na-free acid (pH 3.7-3.8) solutions. It was 528 +/- 46 for batrachotoxin-modified sodium channels. Modified channels were less sensitive to hydrogen block as compared with normal ones. The difference in apparent pKa for acid group between normal and modified channels was about 0.40.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Modulation of sodium currents in rat dorsal root ganglion neurons by sulfur dioxide derivatives

The effect of sulfur dioxide (SO2) derivatives, a common air pollutant and exists in vivo as an equilibrium between bisulfate and sulfite, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in cultured post-natal dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. SO2 derivatives on two types of sodium currents were either inhibitory or stimulatory depending on the kinetic parameters tested. At a holding potential of -80 mV, SO2 derivatives suppressed TTX-S sodium currents when depolarizing potential was negative to -30 mV and TTX-R sodium currents when negative to -10 mV but they increased them when the depolarizing potential was positive to -30 or -10 mV. SO2 derivatives shifted the conductance-voltage curve for TTX-R sodium currents in the depolarizing direction but had little effect on that for TTX-S sodium currents. The steady-state inactivation curve for TTX-R sodium channel was shifted by SO2 derivatives in the depolarizing direction as that for TTX-S sodium channel. SO2 derivatives changed the reversal potential and increased the maximum conductance of two types of sodium channels. SO2 derivatives postponed the activating time and delayed the inactivation of sodium currents. The results suggest that SO2 derivatives would increase the excitability of neurons and alter the ion selectivity for two types of sodium currents.     

Hydrogen ion currents through sodium channels in myelinated nerve fiber membranes

Ionic currents in the nodal membrane of myelinated frog nerve fibre were measured under voltage clamp conditions when the Ranvier node was bathed in solutions containing impermeant cations instead Na. At pH lower than 4.0 small (less than 0.1 nA) currents were detected which rose to peak and then decayed more slowly. Kinetics and voltage range of activation of these currents were similar to those of usual sodium currents at low pH. These currents were reversibly blocked by benzocaine (1 mM). All this permitted identifying them as currents through sodium channels. Experiments in which concentrations of substituting cations (tris+, choline+), Ca2+ and H+ ions were varied showed that the inward currents observed are carried by hydrogen (or hydronium) ions. According to reversal potential measurements the relative permeability of the channels (PH/PNa) is equal to 203 +/- 14 on the average. It is concluded that the energy barriers for H+ in sodium channel are much lower than for Na+, but their passage through the channel is slow because of binding to an acidic group in the channel.     

Hydrogen currents through aconitine-modified sodium channels in nerve fiber membranes

Ionic currents in nodal membrane treated with aconitine were measured under voltage clamp conditions when nodes were bathed in Na-free solutions. At pH lower than 4.6 inward ionic currents were detected which had kinetics and voltage range of activation analogous to those of aconitine-modified sodium channels at low pH. These currents were blocked by benzocaine (2 mM). Experiments with various concentrations of Ca2+, tris+, TEA+, choline+ ions showed that these ions are essentially impermeable both at normal and acidic pH. It is concluded that the inward currents observed are carried by H+ (or H3O+) ions through aconitine-modified sodium channels. From reversal potential measurements relative permeability (PH/PNa) of sodium channels is estimated to be 1059 +/- 88. The results suggest that the aconitine-modified channel is a rather wide water-filled pore and the rate of H+ passing through the channel is limited by its binding to an acidic group.     

Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels

We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.     

Stimulus-dependent blockade of node of Ranvier sodium channels by ethmozine

The effect of antiarrhythmic drug ethmozine on sodium channels in Ranvier node was studied by the voltage clamp technique. Both outside and inside application of ethmozine induced a decrease of sodium current I Na, the time course of I Na and the sodium inactivation being unchanged. The ethmozine-induced Na channel blockade induced tonic (stationary) and phasic (transient stimulus-dependent) components. The tonic blockade of I Na developed slowly and could be accelerated by frequent electric stimulation of the membrane. The phase dependent blockade became more profound with an increase in the pulse rate or amplitude of depolarizing pulses. The prolonged (1 s) membrane depolarization did not bring about an additional blockade of I Na. It is concluded that the phasic blockade is due to the interaction of ethmozine with open Na channels. The noninactivating batrachotoxin-modified Na channels were insensitive to ethmozine. It is found that the increase in outside potassium concentration from 2.5 to 20mM induced both a decrease of the tonic blockade and an increase of the phasic one. The possible nature of the ionic blockade is discussed. The effect of ethmozine is compared with that of tertiary and quaternary local anesthetics.     

Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Density ratios of inward currents

Correlations between densities of different types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three age groups of rats (5-9 days, 45 days and 90 days postnatally). A linear dependence between the densities of high-threshold calcium and slow sodium currents was found. No correlation was observed between the densities of different inward currents in neurons with low-threshold calcium inward current. An inverse dependence was observed between the densities of transmembrane currents in cells having only two types of channels ("fast" sodium and high-threshold calcium ones). Neurons exhibiting slow TTX-resistant sodium and/or low-threshold calcium channels did not show inverse dependence between the densities of "fast" sodium and high-threshold calcium currents.     

Use of the single electrode voltage clamp to perform noise and relaxation studies of acetylcholine-activated channels in Aplysia neurons

The single electrode voltage clamp has been used to perform fluctuation analysis ("noise" analysis) and relaxation experiments in order to study the average lifetime and conductance of ACh-activated sodium channels in Aplysia neurons. Measured values of average channel lifetime, which is approximately 20 msec at --80 mV and 11 degrees C, and elementary conductance, which is approximately 8 pS, are consistent with previously published results using two electrode clamping. The frequency response of the clamp was evaluated to determine its capabilities and limitations for the study of membrane currents. Sinusoidal currents injected into a voltage-clamped model membrane to simulate the frequency components of membrane noise are accurately reproduced at frequencies up to 500 Hz. Following a voltage clamp command, the new membrane potential is established in less than 2 msec, and current relaxations recorded after that time can be used to determine average channel lifetime. Since the frequency response of the clamp is much greater than the average lifetime of ACh-activated channels in Aplysia neurons, the single electrode voltage clamp is comparable to conventional two-electrode systems for investigating the properties of these channels, and may also be useful in other systems in which the time course of membrane currents is much slower than the frequency response of the clamp.     

Do neurons have a reserve of sodium channels for the generation of action potentials? A study on acutely isolated CA1 neurons from the guinea‐pig hippocampus

The density of voltage-gated sodium channels is high in several regions of the neuronal membrane. It is unclear if this density of channels represents a reserve for the neuron, or if it fulfils a special role in action potential firing. This problem was addressed by studying sodium currents and action potentials in acutely isolated hippocampal CA1 neurons whose number of active sodium channels was acutely changed by applying the sodium channel blocker tetrodotoxin (TTX) at different concentrations. The results show that more than a third of the sodium channels can fail without affecting the single action potential. Thus, the neurons have a remarkable surplus of sodium channels. The surplus, however, is necessary for repetitive action potential firing, as every decrease in the fraction of sodium channels reduces the maximal frequency of action potentials that can be generated by the neuron.     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes

Functional receptor channels activated by GABA and other neurotransmitters were "transplanted" from rat brain to Xenopus oocytes by injecting the oocytes with total poly(A)+ mRNA isolated from rat or chick brain. Membrane currents elicited in the oocyte by GABA inverted polarity at about the chloride equilibrium potential (ca. -25 mV). Pentobarbital potentiated the GABA-activated currents, without appreciably changing the reversal potential or form of the current-voltage relationship. At low (less than 10(-5) M) concentrations of GABA, pentobarbital (100 microM) potentiated the responses by a factor of 10 or more, but responses to high (ca. 1 mM) concentrations of GABA were almost unchanged. Half-maximal activation of the response was obtained with about 3 X 10(-5) M GABA when applied alone and with about 4 X 10(-6) M GABA when applied together with 100 microM pentobarbital. At low doses of GABA, the size of the current increased as the 1.4th power of GABA concentration, but this relationship became nearly linear in the presence of pentobarbital. The potentiation of the GABA response increased linearly with concentrations of pentobarbital up to about 300 microM, reaching a maximum of about 50-fold. At higher concentrations of pentobarbital, the response to GABA declined. Relaxations of GABA-activated currents following voltage steps became slower in the presence of pentobarbital, suggesting that the open life-time of the channels was prolonged. In addition to actions on GABA-activated currents, pentobarbital itself elicited a small membrane current that inverted polarity at a potential (-10 mV) more positive than the GABA-activated current.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below −10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance–voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Properties and rundown of sodium-activated potassium channels in rat olfactory bulb neurons.

We have used single-channel recording techniques to investigate the properties of sodium-activated potassium channels (KNa channels) in cultured rat olfactory bulb neurons, and in large neurons in the mitral cell layer of thin slices of olfactory bulb. Ion channels highly selective for potassium over sodium and chloride, and requiring 10-180 mM internal sodium (Nai) for their activation, were present in approximately 75% of inside-out membrane patches detached from cultured olfactory bulb neurons. Most of these patches contained several KNa channels. KNa channels were seen in cell-attached patches only when Nai was raised by including veratridine in the extracellular medium. Preincubation of the cell in TTX or removal of extracellular sodium prevented this effect of veratridine, confirming that the channels observed under these conditions were indeed KNa channels. Lithium did not substitute for Nai in activating these channels. With 150 mM potassium on both sides of the membrane, KNa channels had a single-channel conductance of 172 pS, and at least two subconducting states were observed in addition to this fully open state. Under these ionic conditions, the channels exhibited linear fully open channel current-voltage curves over the potential range of -100 to 0 mV. At voltages more positive than the potassium equilibrium potential, the single-channel currents exhibited inward rectification as a result of sodium block of outward potassium current. The channels opened in bursts, during which they fluctuated between the fully open and closed states, and the substates. Between bursts they sometimes entered a long-lived inactive state that could last for up to several minutes. In addition, KNa channels in the detached patches exhibited rundown, a progressive irreversible loss in activity, over a time course that varied from less than 1 min to longer than 1 hr. Rundown of KNa channel activity in cell-attached patches (in the presence of veratridine) did not occur, suggesting that some intracellular factor necessary for KNa channel activity is lost when the membrane patch is detached from the cell.     

Properties of endogenous voltage-dependent sodium currents in Xenopus laevis oocytes.

Endogenous voltage-dependent sodium currents were recorded using standard 2-microelectrode techniques in Xenopus laevis oocytes. Maximal inward current occurred at -10 mV with an average amplitude of -279 +/- 17 nA and steady-state inactivation was half-maximal at a voltage of -38 +/- 0.5 mV. Currents were blocked by low concentrations of tetrodotoxin (TTX) with an IC50 value of 6 nM. These properties make the endogenous sodium current in Xenopus oocytes similar to sodium currents expressed following injection of mammalian brain RNA. While endogenous sodium channels have the potential to complicate analysis when using the oocyte expression system, they are only present at significant levels in rare batches of oocytes (less than 5%). Our results do stress the need, however, to reproduce results from exogenous expression studies across several batches of oocytes from different donors.     

Interactions of the pyrethroid fenvalerate with nerve membrane sodium channels: temperature dependence and mechanism of depolarization

Depolarization of nerve membranes is an important component of the mode of action of pyrethroids, and its negative temperature dependence parallels that of insecticidal activity. We studied the mechanism and temperature dependence of depolarization of crayfish giant axons by pyrethroids, using intracellular microelectrode and voltage clamp techniques. Membrane depolarization caused by tetramethrin and fenvalerate was greater at 10 degrees C than at 21 degrees C, and was reversible upon changing the temperature. Short-duration depolarizing pulses in voltage-clamped fenvalerate-treated axons induced prolonged sodium currents that are typical of other pyrethroids, but the decay of the tail current following repolarization was extremely slow, lasting several minutes at the large negative holding potential of -120 mV. At the normal resting potential, the tail current did not decay completely, and even without stimulation, a steady-state sodium current developed, which could account for the depolarization. The steady-state current induced by fenvalerate at the resting potential was much larger at 8 degrees C than at 21 degrees C, accounting for the negative temperature dependence of the depolarization. The negative temperature dependence of the steady-state current seems to be due ultimately to the great stabilizing effect of low temperature on the open-modified channel. When the steady-state current was induced at the resting potential, hyperpolarization to more negative potentials caused it to decay with exactly the same time course as tail currents induced by short-duration depolarizing pulses, indicating that both types of currents are carried by identically-modified channels. The modified channels were shown to be inactivated very slowly at potentials more positive than - 100 mV, accounting for the limited depolarization observed in micro-electrode experiments. Even when applied directly to the internal face of the membrane, the effect of fenvalerate on the sodium channel developed slowly, taking more than 90 min to reach its final level. Fenvalerate did not significantly affect potassium currents.