Interaction of tetramethrin and deltamethrin at the single sodium channel in rat hippocampal neurons.

Type I and type II pyrethroids are known to modulate the sodium channel to cause persistent openings during depolarization and upon repolarization. Although there are some similarities between the two types of pyrethroids in their actions on sodium channels, the pattern of modification of sodium currents is different between the two types of pyrethroids. In the present study, interactions of the type I pyrethroid tetramethrin and the type II pyrethroid deltamethrin at rat hippocampal neuron sodium channels were investigated using the inside-out single-channel patch clamp technique. Deltamethrin-modified sodium channels opened much longer than tetramethrin-modified sodium channels. When 10 microM tetramethrin was applied to membrane patches that had been exposed to 10 microM deltamethrin, deltamethrin-modified prolonged single sodium currents disappeared and were replaced by shorter openings which were characteristic of tetramethrin-modified channel openings. These single-channel data are compatible with previous whole-cell competition study between type I and type II pyrethroids. These results are interpreted as being due to the displacement of the type II pyrethroid molecule by the type I pyrethroid molecule from the same binding site or to the allosteric interaction of the two pyrethroid molecules at separate sodium channel sites.     

Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels

The differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) single sodium channel currents in rat dorsal root ganglion (DRG) neurons were investigated using the outside-out configuration of patch-clamp technique. Channel conductances were 10.7 and 6.3 pS for TTX-S and TTX-R sodium channels, respectively, at a room temperature of 24–26°C. The single-channel current of TTX-S sodium channels at the test potential of −30 mV was −1.27 ± 0.25 pA, and was not changed after exposure to 10 μM tetramethrin (−1.28 ± 0.23 pA). The open time histogram of TTX-S single-channel currents could be fitted by a single exponential function with a time constant of 1.27 ms. After exposure to 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (τ_fast) and 5.73 ms (τ_low). The percentage of contribution of each component to the population was 62% for the fast component representing the normal channels and 38% for the slow component representing the tetramethrin modified channels. The amplitudc of TTX-R single-channel currents was slightly changed from −0.72 ± 0.14 to −0.83 ± 0.07 pA by 10 μM tetramethrin. The open time histogram of TTX-R single-channel currents could be fitted by a single exponential function with a time constant of 1.92 ms. In the presence of 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (τ_fast) and 9.75 ms (τ_slow). The percentage of contribution of each component was 15% for the fast, unmodified component and 85% for the slow, modified component. Differential effects of tetramethrin on the open time distribution of single sodium channel currents explains the differential sensitivity of TTX-S and TTX-R sodium channels.     

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 tetramethrin, fenvalerate and DDT at the sodium channel in rat dorsal root ganglion neurons

Type I and type II pyrethroids and dichlorodiphenyltrichloroethane (DDT) are known to modulate the sodium channel to cause the hyperexcitatory symptoms of poisoning in animals. However, since the degrees to which neuronal sodium channel parameters are altered differ, a question is raised as to whether these insecticides bind to the same site in the sodium channel. Competition patch-clamp experiments were performed using rat dorsal root ganglion neurons which are endowed with tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.d-trans-Tetramethrin,S,S-fenvalerate andp,p′-DDT caused a slowly rising and slowly falling tail current o to be developed in tetrodotoxin-sensitive sodium channels. In tetrodotoxin-resistant sodium channels, these insecticides, particularly tetramethrin and fenvalerate, generated a large and prolonged tail current upon repolarization. The effects of tetramethrin were reversible after washing with drug-free solution, whereas the effects of fenvalerate and DDT were irreversible. When fenvalerate application was followed by tetramethrin application, the characteristic changes in current by fenvalerate disappeared and the characteristic changes by tetramethrin appeared. After washout, the characteristic current pattern of fenvalerate reappeared. These results can be explained by assuming that the tetramethrin molecule displaces the fenvalerate molecule from the same binding site in the sodium channel protein, or that tetramethrin and fenvalerate bind to separate sodium channel sites which interact allosterically with each other. DDT interacted with fenvalerate and tetramethrin in the same manner.     

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.     

Chemical modification of sodium channel inactivation: separate sites for the action of grayanotoxin and tetramethrin

The gating mechanisms of the sodium channel are known to be modified by grayanotoxin and the pyrethroid tetramethrin. Voltage clamp experiments with internally perfused squid giant axons were performed to determine whether or not these two chemicals shared a common site of action in exerting their effects. An additive effect of the two drugs in prolonging sodium currents was observed. Additionally, the characteristic tetramethrin-induced sodium tail current and the grayanotoxin-induced hyperpolarizing shift in the voltage that activated the sodium current were observed simultaneously and independently of the order of drug introduction. Inactive stereoisomers of tetramethrin, which are known to prevent the active tetramethrin stereoisomers from exerting their effect, had no effect on the development of the grayanotoxin-induced modifications of sodium current. It was concluded that tetramethrin and grayanotoxin act at separate sites of action in modifying the sodium channel gating mechanisms in the squid axon membrane.     

Ion permeation and selectivity of squid axon sodium channels modified by tetramethrin

The pyrethroid tetramethrin greatly prolongs the sodium current during step depolarization and the sodium tail current associated with step repolarization of the squid axon membrane. Non-linear current-voltage relationships for the sodium tail current were analyzed to assess the open sodium channel properties which included the permeation of various cations, calcium block and cation selectivity. Tetramethrin had no effect on any of these properties. It was concluded that tetramethrin modifies the sodium channel gating machinery without affecting the pore properties.     

Steroidal alkaloid batrachotoxin--instrument for studying voltage-regulated sodium channels

Results of recent studies on the batrachotoxin (BTX) effect on the properties of voltage-operated sodium channels in excitable membranes are summarized in the review. The following problems are considered: allosteric interaction of the BTX receptor with structural entities of the sodium channel responsible for its activation, inactivation, ion selectivity, binding of polypeptide (scorpion and anemone) toxins, local anesthetics and many blocking drugs; relationship between BTX-induced changes in the sodium conductance and intramembrane charge movement; relationship between ion selectivity and effective pK of the selectivity filter acid group of sodium channels modified by BTX or aconitine; effects of BTX on the behaviour and conductance (gamma) of single sodium channels. The problem of the BTX receptor location and possible mechanism of the sodium channel modification by BTX are discussed.     

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.     

Interaction of the pyrethroid insecticides tetramethrin and cypermethrin with enteric cholinergic transmission in the guinea-pig

In electrically-stimulated longitudinal muscle-myenteric plexus preparations of the guinea-pig ileum, the Type I pyrethroid insecticide tetramethrin (1-100 microM) caused a biphasic response consisting of an early transient increase followed by a sustained decrease in the amplitude of cholinergic contractions. The cholinergic potentiation was antagonized by phenytoin (3 microM), which also prevented the increase in twitch height caused by veratridine (30 nM). The late inhibitory effect of tetramethrin probably involved a direct action on the musculature since contractile responses to applied acetylcholine (100 nM) or histamine (300 nM) were also depressed by this compound. Cypermethrin (1-100 microM), a Type II pyrethroid, had only a minor enhancing effect on electrically evoked contractions. Cypermethrin (30, 60 microM), but not tetramethrin, antagonized the cholinergic response induced by the GABA-A receptor agonist 3-aminopropane sulphonic acid (1-100 microM). These results suggest that neural Na+ channels activation may underlie pyrethroid-induced potentiation of enteric cholinergic transmission. In small intestine, however, cypermethrin is also effective as a noncompetitive antagonist of GABA-A receptor mediated cholinergic contractions.     

Pharmacologic analysis of sodium channel inactivation in a nerve fiber membrane

Recent advances in the study of the effects of various enzymes, toxins, drugs and ions on Na channels inactivation are reviewed. The available data suggest the protein "inactivation subunit" (IS) to span the membrane. The internal end of this IS protrudes from the membrane to the axoplasm and acts as an inactivation gate (h-gate). It can be affected by intracellular application of some proteases (endopeptidases), protein-specific reagents and drugs, removing (completely or partially) the fast sodium inactivation. The ultraslow sodium inactivation, resistant to proteases, is apparently due to the conformational changes of that part of the channel which is buried in the membrane lipid matrix. The outer end of the IS is provided with chemical groups having a high affinity to anemone and scorpion toxins inducing the modification of Na inactivation when applied from outside the fibre. Batrachotoxin and aconitine cause a simultaneous modification of inactivation, activation and selectivity of Na channels by interacting with a single "receptor site" of the channel. It is proposed that this receptor is disposed in the hydrophobic part of the channel and is allosterically linked with different subunits responsible for the principal channel functions. It is tempting to assume that the batrachotoxin receptor belongs to that subunit which plays a key role in the normal structural interaction of various channel subunits. Inactivation process is critically involved in the voltage- and frequency-dependent inhibition of sodium currents by various quaternary and tertiary amines among which there are local anesthetics and antiarrhythmics.     

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.     

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.     

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.     

The permeation and activation properties of brain sodium channels change during development

BTX-modified sodium channels from 15-day embryonic (E15) rat forebrains were studied in planar lipid bilayers. Compared to postnatal sodium channels, E15 channels had a lower maximal single channel conductance, whereas their permeation pathway sensed a comparable surface charge density and had a similar apparent binding affinity for sodium ions. The steady-state activation curve of E15 channels was significantly more hyperpolarized and had a shallower slope than postnatal channels. The apparent BTX binding affinity was significantly lower for E15 channels than for postnatal channels. Finally, E15 channel alpha-subunits displayed a lower apparent molecular weight, and a lower sialylation level than postnatal sodium channel alpha-subunits. Together with previous studies, our data suggested that the observed functional differences between E15 and postnatal voltage-dependent sodium channels cannot be explained solely by the observed differences in channel sialylation, and hence they also appeared to reflect the presence of other channel structural differences.     

Modification of the gating mechanism of incurrent sodium channels by an osmotic pressure gradient on a perfused neuron

The influence of the osmotic pressure gradient was investigated by the voltage clamp method. It was shown that the osmotic gradient changed both activation and inactivation of the sodium conductivity and decreased the sodium current amplitude. Conductivity of single sodium channels was not affected by the osmotic pressure gradient. It is concluded that the decrease in sodium current amplitude is due to a decrease in the number of open channels.     

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effect of external pH on the amplitude of currents through single sodium channels in cultured mouse neuroblastoma cells C 1300, clone N18A-1 was studied. Currents through single sodium channels in outside-out membrane patches were measured at normal (7.2) and low (5.4) pH of the external solution. With a decrease of the external pH to 5.4, about two-fold reversible reduction of the amplitude of single sodium channel currents (at testing potentials of -10-30 mV) was observed. The data obtained confirm the suggestion that the inhibition of macroscopic sodium currents with lowering of pH of the extracellular solution is due to the decrease in the ionic current flowing through single open channels.     

Features of the kinetic and stationary characteristics of aconitine-modified sodium channels

Kinetic and steady-state characteristics of aconitine-modified sodium channels were studied in the Ranvier node membrane. Aconitine-modified sodium channels are shown to be inactivated only partially. The voltage dependence of the fraction of noninactivated channels (h infinity) may be described by a three-state model of the channel with closed, open and inactivated states. A reasonable agreement with the data was obtained when parameters of the inactivated state were supposed to be not changed after aconitine modification of the channels. The membrane repolarization to -70 divided by -110 mV, after long (10 ms) depolarizing shift induces firstly fast current decay ("tail") and then its rather slow increase to a steady-state level. Kinetics of this current requires two or more open states to be postulated.     

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.     

Chaotic spiking in the Hodgkin-Huxley nerve model with slow inactivation of the sodium current

The Hodgkin-Huxley (HH) equations with a modification in which the inactivation process (h variable) of sodium channels is slightly slowed down are investigated. It is shown that this slight modification changes the HH dynamics to a completely different one, with chaotic spiking and very long interspike intervals appearing in a generic manner, although the initiation mechanism of repetitive firing is a simple Hopf bifurcation.