Interactions of pyrethroids and octylguanidine with sodium channels of squid giant axons

Kinetics of pyrethroid-modified sodium channels and the interaction of N-octylguanidine with the modified channels have been studied with internally perfused and voltage-clamped squid giant axons. The pyrethroids used were 1R-cis-phenothrin; 1R-cis-permethrin; 1R-cis-cyphenothrin; and 1R-cis-deltamethrin. Modification of sodium channels by pyrethroids resulted in marked slowing of opening and closing kinetics. The rate at which sodium channels arrived at the open pyrethroid-modified state during a depolarizing step was independent of the concentration of pyrethroids applied. The time of exposure to pyrethroids required for the pyrethroid-induced sodium tail current following a step depolarization to reach a steady-state amplitude was independent of the frequency of short (5 ms) depolarizing pulses, and in the pronase-treated axons was independent of the membrane potential (0 mV or -90 mV). We conclude that sodium channels are modified by pyrethroids primarily in the closed resting state. A small fraction of sodium channels is modified in the open state. The dose-response curve for N-octylguanidine block of sodium channels was not shifted by pyrethroids. The rate at which the pyrethroid-modified sodium channels were blocked by octylguanidine during a depolarizing step depended neither on the concentration of pyrethroids nor on the depolarizing potential, but depended on the concentration of octylguanidine. The time course of the pyrethroid-induced slow sodium tail current was not altered by octylguanidine. We conclude that the actions of pyrethroids and N-octylguanidine on sodium channels are independent of each other.     

Sodium depletion in the periaxonal space of the squid axon treated with pyrethroids

Pyrethriods are known to increase the steady-state sodium current during a step depolarization and to increase and prolong the tail sodium current associated with a step repolarization of the membrane. The pyrethroid-induced tail sodium current of squid axons developed as a function of the duration of the conditioning depolarizing pulse. However, with further lengthening the conditioning pulse duration, it decreased, further increased, or remained constant depending on the direction of sodium current during the depolarization, irrespective of the membrane potential per se. The depletion or accumulation of sodium in the periaxonal space during a 200-ms conditioning depolarizing pulse in the axon internally treated with pronase, pyrethroids, or both, was demonstrated by measurements of the changes in sodium reversal potential. Thus the observed changes in tail current amplitude as a function of the conditioning pulse duration are explicable in terms of changes in sodium concentration in the periaxonal space without assuming inactivation of the pyrethroid-modified channel.     

Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells

The effects of 4 different pyrethroid insecticides on sodium channel gating in internally perfused, cultured mouse neuroblastoma cells (N1E-115) were studied using the suction pipette, voltage clamp technique. Pyrethroids increased the amplitude of the sodium current, sometimes by more than 200%. Activation of the sodium current occurred at more hyperpolarized potentials than under control conditions. The declining phase of the sodium current during depolarization was markedly slowed down and after repolarization of the membrane a large, slowly decaying sodium tail current developed. Pyrethroids did not affect the sodium current reversal potential, steady-state sodium inactivation or recovery from sodium channel inactivation. The amplitude of the pyrethroid-induced slow tail current was always proportional to the sodium current at the end of the preceding depolarizing pulse. The rate of decay of the slow tail current strongly depended on pyrethroid structure and increased in the order deltamethrin, cyphenothrin, fenfluthrin and phenothrin. The rate of decay further depended on membrane potential and temperature. Below -85 m V the instantaneous current-voltage relationship of the slow tail current showed a negative slope conductance. The tail current decayed more slowly at low temperatures. Arrhenius plots indicated that the relaxation of open sodium channels to a closed state involved a higher energy barrier for pyrethroid-affected than for normal channels. The energy barrier was higher after deltamethrin than after the non-cyano pyrethroid fenfluthrin. It is concluded that in mammalian neuronal membrane pyrethroids selectively reduce the rate of closing of sodium channels both during depolarization and after repolarization of the nerve membrane.     

A use-dependent sodium current modification induced by type I pyrethroid insecticides in honeybee antennal olfactory receptor neurons

We studied the mode of action of type I pyrethroids on the voltage-dependent sodium current from honeybee olfactory receptor neurons (ORNs), whose proper function in antenna is crucial for interindividual communication in this species. Under voltage-clamp, tetramethrin and permethrin induce a long lasting TTX-sensitive tail current upon repolarization, which is the hallmark of an abnormal prolongation of the open channel configuration. Permethrin and tetramethrin also slow down the sodium current fast inactivation. Tetramethrin and permethrin both bind to the closed state of the channel as suggested by the presence of an obvious tail current after the first single depolarization applied in the presence of either compounds. Moreover, at first sight, channel opening seems to promote tetramethrin and permethrin binding as evidenced by the progressive tail current summation along with trains of stimulations, tetramethrin being more potent at modifying channels than permethrin. However, a use-dependent increase in the sodium peak current along with stimulations suggests that the tail current accumulation could also be a consequence of progressively unmasked silent channels. Experiments with the sea anemone toxin ATX-II that suppresses sodium channels fast inactivation are consistent with the hypothesis that these silent channels are either in an inactivated state at rest, or that they normally inactivate before they open so that they do not participate to the control sodium current. In honeybee ORNs, three processes lead to a use-dependent pyrethroid-induced tail current accumulation: (i) a recruitment of silent channels that produces an increase in the peak sodium current, (ii) a slowing down of the sodium current inactivation produced by prolongation of channels opening and (iii) a typical deceleration in current deactivation. The use-dependent recruitment of silent sodium channels in honeybee ORNs makes pyrethroids more potent at modifying neuronal excitability.     

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.     

State-dependent block of rat Nav1.4 sodium channels expressed in xenopus oocytes by pyrazoline-type insecticides

Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Na(v)1.4 sodium channels at a membrane potential of -120 mV, but depolarization to -60 mV or -30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.     

Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+β1+β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+β1+β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.     

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.     

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(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.     

Modulation of nerve membrane sodium channel activation by deltamethrin.

Deltamethrin is a highly potent pyrethroid insecticide that causes hypersensitivity, choreoathetosis, tremors, and paralysis in mammals. It is known to modify the sodium channel in such a way as to prolong the tail current associated with step repolarization following a depolarizing pulse. Using the axial-wire voltage-clamp technique with the giant axon of the squid Loligo pealei, we have demonstrated that deltamethrin also greatly slows the opening of the sodium channel. This was first observed as a decrease, by as much as 80%, in the peak sodium current flowing during a short, 10 ms depolarization. Current flowing through these slowly opening deltamethrin modified sodium channels was observed during the first depolarizing pulse after deltamethrin exposure and developed with a time constant of 320 ms. This supports the idea that deltamethrin can modify sodium channels when they are in the closed or resting state. Further, evidence of this hypothesis was provided by experiments using 0.1 and 10 microM deltamethrin and measuring the tail current amplitude after depolarizing pulses of varying duration (1-1200 ms). The mean time constant for the increase in tail current amplitude was almost concentration independent; 253 ms at 0.1 microM and 193 ms at 10 microM. We conclude that deltamethrin modifies the activation kinetics of sodium channels in such a way as to slow opening and that this modification occurs predominantly when channels are in the closed or resting state.     

Induction of pseudo-periodic oscillation in voltage-gated sodium channel properties is dependent on the duration of prolonged depolarization

The neuronal voltage-gated sodium channels play a vital role in the action potential waveform shaping and propagation. Here, we report the effects of prolonged depolarization (1-160 s) on the detailed kinetics of activation, fast inactivation and recovery from slow inactivation in the rNa(v)1.2a voltage-gated sodium channel alpha-subunit expressed in Chinese hamster ovary (CHO) cells. Wavelet analysis revealed that the duration and amplitude of a prolonged sustained depolarization altered all the steady state and kinetic parameters of the channel in a pseudo-oscillatory fashion with time-variable period and amplitude, often superimposed on a linear trend. The half steady state activation potential showed a reversible depolarizing shift of 5-10 mV with duration of prolonged depolarization, while half steady state inactivation potential showed a hyperpolarizing shift of 43-55 mV. The time periods for most of the parameters relating to activation and fast and slow inactivation, lie close to 28-30 s, suggesting coupling of these kinetic processes through an oscillatory mechanism. Co-expression of the beta1-subunit affected the time periods of oscillation (close to 22 s for alpha + beta1) in steady state activation parameters. Application of a pulse protocol that mimicked paroxysmal depolarizing shift (PDS), a kind of depolarization seen in epileptic discharges, instead of a sustained depolarization, also caused oscillatory behaviour in the rNav1.2a alpha-subunit. This inherent pseudo-oscillatory mechanism may regulate excitability of the neurons, account for the epileptic discharges and subthreshold membrane potential oscillation and offer a molecular memory mechanism intrinsic to the neurons, independent of synaptic plasticity.     

Differential Effects of Tityus bahiensis Scorpion Venom on Tetrodotoxin-Sensitive and Tetrodotoxin-Resistant Sodium Currents

We examined modification of sodium channel gating by Tityus bahiensis scorpion venom (TbScV), and compared effects on native tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents from rat dorsal root ganglion neurons and cardiac myocytes. In neurons, TbScV dramatically reduced the rate of sodium current inactivation, increased current amplitude, and caused a negative shift in the voltage-dependence of activation and inactivation of tetrodotoxin-sensitive channels. Enhanced activation of modified sodium channels was independent of a depolarizing prepulse. We identified two components of neuronal tetrodotoxin-resistant current with biophysical properties similar to those described for NaV1.8 and NaV1.9. In contrast to its effects on neuronal tetrodotoxin-sensitive current, TbScV caused a small decrease in neuronal tetrodotoxin-resistant sodium current amplitude and the gating modifications described above were absent. A third tetrodotoxin-resistant current, NaV1.5 recorded in rat cardiac ventricular myocytes, was inhibited approximately 50% by TbScV, and the remaining current exhibited markedly slowed activation and inactivation. In conclusion, TbScV has very different effects on different sodium channel isoforms. Among the neuronal types, currents resistant to tetrodotoxin are also resistant to gating modification by TbScV. The cardiac tetrodotoxin-resistant current has complex sensitivity that includes both inhibition of current amplitude and slowing of activation and inactivation.     

The role of altered sodium currents in action potential abnormalities of cultured dorsal root ganglion neurons from trisomy 21 (down syndrome) human fetuses

Trisomy 21 (Down syndrome) results in abnormalities in electrical membrane properties of cultured human fetal dorsal root ganglion (DRG) neurons. Action potentials have faster rates of depolarization and repolarization, with decreased spike duration, compared to diploid neurons. In order to analyze the faster depolarization rate observed in trisomic neurons, we examined sodium currents of cultured human fetal DRG neurons from trisomy 21 and control subjects, using the whole-cell patch-clamp technique. The neurons were replated in culture to reduce dendritic spines. Two components of the sodium current were identified: (1) a fast, tetrodotoxin (TTX)-sensitive current; and (2) a slow, TTX-resistant component. The inactivation curves of both current types in trisomic neurons showed a shift of approximately 10 mV towards more depolarized potentials compared to control neurons. Thus, whereas essetially all of the fast sodium channels were inactivated at normal resting potentials in control neurons, approximately 10% of these channels were available for activation in trisomy 21 cells. Furthermore, the fast current showed accelerated activation kinetics in trisomic neurons. The slow sodium current of trisomic neurons showed slower deactivation kinetics than control cells. No differences were observed between trisomic and control neurons in the maximal conductance or current densities of either fast or slow current components. These data indicate that the greater rate of depolarization in trisomy 21 neurons at resting potentials is primarily due to activation of residual fast sodium channels that also have a faster time course of activation.     

Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids

Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding.     

Differential sensitivity of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels to the insecticide allethrin in rat dorsal root ganglion neurons

The pyrethroid insecticides are known to modify neuronal sodium channels to cause a prolongation of whole cell current. The sodium channels expressed in the dorsal root ganglion neurons of the rat are of two types, one highly sensitive to tetrodotoxin and the other highly resistant to tetrodotoxin. The pyrethroid allethrin exerted profound effects on tetrodotoxin-resistant sodium channels while causing minimal effects on tetrodotoxin-sensitive sodium channels. Currents derived from tetrodotoxin-resistant sodium channels were greatly prolonged during a step depolarization; the tail currents upon repolarization were also augmented and prolonged. In the tetrodotoxin-sensitive sodium channel currents, these changes caused by allethrin were much smaller or negligible. The activation and inactivation voltages of tetrodotoxin-resistant peak sodium currents were not significantly altered by allethrin. The differential action of allethrin on the two types of sodium channels would be important not only in identifying the target molecular structure but also in interpreting the symptoms of poisoning in mammals.     

Activation and inactivation of batrachotoxin-modified sodium channels of nerve fiber membranes in the frog

Currents through batrachotoxin (BTX)-modified sodium channels in frog myelinated nerve were measured under voltage-clamp conditions. Nonlinearity of "instantaneous" current-voltage relations was taken into account when determining steady-state parameters of channel activation. BTX induces the shift of voltage dependence of channel activation towards more negative potentials by 67 mV, without changes in its steepness. Current kinetics and effect of preceding depolarization on current size suggest that BTX-modified channels are capable for partial inactivation. High level of steady-state conductance of BTX-modified channels can be explained by suggestion that open state of the channel is energetically more profitable than inactivated one. It is concluded that effect of BTX on inactivation is different in principle from that of pronase and protein reagents.     

Modification of sodium channel kinetics by the insecticide tetramethrin in crayfish giant axons

The effects of the pyrethroid insecticide tetramethrin on voltage-dependent sodium channels were studied with internally perfused crayfish giant axons. At low concentrations in the order of 10-8-10-9M, tetramethrin caused an increase in depolarizing after-potential which in turn triggered repetitive after-discharges. Under Voltage clamp conditions, the sodium current was markedly prolonged during a step depolarization, and a large and prolonged sodium tail current appeared upon step repolarization. A population of sodium channels having activation and inactivation kinetics identical to those in control axons was observed in the tetramethrin-poisoned axons, indicating that only a fraction of the channels was modified. The modified channels exhibited remarkably slow kinetics, activating with a time course of 100 msec to 2 sec and inactivating with a time course of 1-5 sec depending on the membrane potential. The voltage dependence of the modified channels was shifted in the direction of hyperpolarization by about 10-20 mV with respect to normal sodium channels. The large inward sodium tail current associated with step repolarization of the membrane decayed with a time course of 20-600 msec. A kinetic hypothesis describing the behavior of sodium channels in a tetramethrin-poisoned axon is presented and discussed in relation of the behavior of the sodium channels modified by other toxins.     

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

Kindling in the cortical nucleus of the amygdala

Adenosine 5'-triphosphate (ATP) produced a long-lasting depolarization in bullfrog spinal ganglion cells. Since the ATP-induced slow depolarization was associated with an increase in membrane resistance and a reverse in polarity (about--90 mV) which was most likely brought about by an inactivation of membrane potassium conductance. In some cells, a rapid and transient depolarization followed by the long-lasting depolarization was produced by ATP and it was markedly reduced in sodium-free solution. ATP reversibly augmented the GABA-induced depolarization which was caused by ionophoresis of GABA. These observations were confirmed using a voltage clamp method. Dose-response analysis of the action of ATP on the GABA-induced response suggests that the facilitatory action of ATP on the GABA response is effected on the GABA receptor channel complexes without changing the GABA affinity.