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.     

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.     

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.     

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.     

Modification of single sodium channels by the insecticide tetramethrin

(+)-trans-Tetramethrin, a pyrethroid insecticide, markedly prolongs the open time of single sodium channels recorded by the gigaohm-seal voltage clamp technique in a membrane patch excised from the N1E-115 neuroblastoma cell. Single channel conductance is not altered by tetramethrin. The modification by tetramethrin occurs in an all-or-none manner in a population of sodium channels. The observed tetramethrin-induced modification of single sodium channels is compatible with previous sodium current data from axons.     

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.     

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.     

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.     

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.     

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.     

The action of pyrethroids on sodium channels in myelinated nerve fibres and spinal ganglion cells of the frog

The interaction of pyrethroids with the voltage-dependent sodium channel was studied in voltage-clamped nodes of Ranvier and isolated spinal ganglion neurons of the clawed frog, Xenopus laevis. In the node, pyrethroids prolonged the sodium tail current associated with a step repolarization of the membrane. It was found that the amplitude of the slow, pyrethroid-induced, sodium tail current (PIT) first increased and then decreased as a function of the duration of membrane depolarization (to -5 mV). This decrease of the PIT amplitude was absent when depolarizations to the sodium equilibrium potential (+40 mV) were used. Measurements of changes in sodium reversal potential indicated that sodium ion depletion in the perinodal space is largely responsible for the inactivation of the pyrethroid-modified sodium current. Inactivation is not completely abolished by pyrethroid treatment since the probability of channel opening, measured in membrane patches excised from spinal ganglion cells, decreased slowly during prolonged depolarization. Analysis of unitary currents indicated that both activation and inactivation are retarded by pyrethroids. The arrival of sodium channels in the pyrethroid-modified open state followed a time course that was slower than both activation and inactivation of unmodified sodium channels. Our findings indicate that sodium channels are modified when in the closed resting state and that both opening and closing kinetics are delayed by pyrethroids.     

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.     

Block of deltamethrin-modified sodium current in cultured mouse neuroblastoma cells: local anesthetics as potential antidotes

Effects of local anesthetics and anticonvulsants on the pyrethroid-modified sodium current in cultured mouse neuroblastoma cells have been investigated using the suction pipette voltage clamp technique. In the presence of 10 microM of the pyrethroid deltamethrin the sodium current consists of an enhanced peak current during membrane depolarization and a slowly decaying, deltamethrin-induced tail current remaining after repolarization. At the onset of block the local anesthetics tetracaine, lidocaine and QX 314 reduced the deltamethrin-induced tail current more effectively than the peak current. Lidocaine, but not phenytoin, caused a time-dependent block of tail currents evoked by membrane depolarizations lasting 10-1000 ms. Both lidocaine- and phenytoin-induced blocks were independent of the membrane potential during the tail current. The anticonvulsants phenytoin, phenobarbital and valproate blocked the tail and the peak sodium current to the same extent, but diazepam, mephenesin and urethane blocked the peak current more effectively. Vitamin E, which suppresses pyrethroid-induced paresthesia of the skin, had no effect on the voltage-dependent sodium current. It is concluded that indirect effects of anticonvulsants on pyrethroid-induced toxic symptoms predominate, whereas local anesthetics preferentially block the pyrethroid-induced tail current. Therefore, local anesthetics are potentially useful pyrethroid antidotes.     

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.     

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.     

Kinetics of sodium current decay during normal axon membrane repolarization and in the presence of scorpion toxin

Decay of sodium currents in repolarization ("tail current") was studied in from axonal membrane. The decay in the membrane repolarization to -40 divided by -60 mV has two exponential components: fast and slow. The fraction of the slow component in the total "tail current" (theta M) decreases as the repolarization potential (Vp) becomes more negative; at Vp more negative than -80 mV "tail" follows practically one-exponential time course. When lengthening the test pulse (at the given Vp) the fraction of the fast component in the "tail" decreases quicker than that of the slow component, following approximately the kinetics of inactivation during the tests pulse. Scorpion toxin treatment results in slowing down "tail" kinetics mainly at the expense of increasing the fraction of the slow component. A kinetic diagram assuming two open state for the channel is suggested. A hypothesis is advanced that scorpion toxin, DDT and trinitrophenol have a common "site" to interact with the gating mechanism of the sodium channel.     

The effect of α-, β adrenergic receptor agonists and antagonists on the efflux of Na and uptake of K by rat brain cortical slices

The effects of norepinephrine on ion fluxes in rat brain cortical slices have now been ascertained. 22Na efflux and 42K influx are enhanced by norepinephrine. The increase in ion fluxes can be blocked by ouabain, phentolamine and propranolol, suggesting that the catecholamine activates a membrane sodium pump by a receptor-mediated step. The facilitation of 22Na efflux is stereospecific as demonstrated by the very weak action of D-norepinephrine at 10(-5) M concentration. Various alpha-adrenergic and beta-adrenergic receptor agonists, including oxymetazoline, naphazoline, clonidine, tramazoline, methoxamine, phenylephrine, L-isoproterenol and methoxyphenamine are potent stimulants of the sodium pump as demonstrated by their enhancement of ion fluxes in rat brain cortical slices. Our results are consistent with the hypothesis that norepinephrine hyperpolarizes central neurons by activating an ouabain-sensitive, receptor-mediated sodium pump.     

Analysis of distinct short and prolonged components in rebound spiking of deep cerebellar nucleus neurons

Deep cerebellar nucleus (DCN) neurons show pronounced post-hyperpolarization rebound burst behavior, which may contribute significantly to responses to strong inhibitory inputs from cerebellar cortical Purkinje cells. Thus, rebound behavior could importantly shape the output from the cerebellum. We used whole-cell recordings in brain slices to characterize DCN rebound properties and their dependence on hyperpolarization duration and depth. We found that DCN rebounds showed distinct fast and prolonged components, with different stimulus dependence and different underlying currents. The initial depolarization leading into rebound spiking was carried by hyperpolarization-activated cyclic nucleotide-gated current, and variable expression of this current could lead to a control of rebound latency. The ensuing fast rebound burst was due to T-type calcium current, as previously described. It was highly variable between cells in strength, and could be expressed fully after short periods of hyperpolarization. In contrast, a subsequent prolonged rebound component required longer and deeper periods of hyperpolarization before it was fully established. We found using voltage-clamp and dynamic-clamp analyses that a slowly inactivating persistent sodium current fits the conductance underlying this prolonged rebound component, resulting in spike rate increases over several seconds. Overall, our results demonstrate that multiphasic DCN rebound properties could be elicited differentially by different levels of Purkinje cell activation, and thus create a rich repertoire of potential rebound dynamics in the cerebellar control of motor timing.     

Characterisation of hyperpolarization-activated currents (I(h)) in the medial septum/diagonal band complex in the mouse

Hyperpolarization-activated cyclic nucleotide gated (HCN) channel subunits are distributed widely, but selectively, in the central nervous system, and underlie hyperpolarization-activated currents (I(h)) that contribute to rhythmicity in a variety of neurons. This study investigates, using current and voltage-clamp techniques in brain slices from young mice, the properties of I(h) currents in medial septum/diagonal band (MS/DB) neurons. Subsets of neurons in this complex, including GABAergic and cholinergic neurons, innervate the hippocampal formation, and play a role in modulating hippocampal theta rhythm. In support of a potential role for I(h) in regulating MS/DB firing properties and consequently hippocampal neuron rhythmicity, I(h) currents were present in around 60% of midline MS/DB complex neurons. The I(h) currents were sensitive to the selective blocker ZD7288 (10 microM). The I(h) current had a time constant of activation of around 220 ms (at -130 mV), and tail current analysis revealed a half-activation voltage of -98 mV. Notably, the amplitude and kinetics of I(h) currents in MS/DB neurons were insensitive to the cAMP membrane permeable analogue 8-bromo-cAMP (1 mM), and application of muscarine (100 microM). Immunofluoresence using antibodies against HCN1, 2 and 4 channel subunits revealed that all three HCN subunits are expressed in neurons in the MS/DB, including neurons that express the calcium binding protein parvalbumin (marker of fast spiking GABAergic septo-hippocampal projection neurons). The results demonstrate, for the first time, that specific HCN channel subunits are likely to be coexpressed in subsets of MS/DB neurons, and that the resultant I(h) currents show both similarities, and differences, to previously described I(h) currents in other CNS neurons.     

Effects of lysophosphatidic acid on sodium currents in rat dorsal root ganglion neurons

Lysophosphatidic acid (LPA), a simple phospholipid, induces pain. To elucidate an involvement of ion channel mechanism in the LPA-induced pain, its effects on sodium currents in rat dorsal root ganglion (DRG) neurons were investigated. LPA suppressed tetrodotoxin-sensitive (TTX-S) sodium current, but increased tetrodotoxin-resistant (TTX-R) sodium current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. In both types of currents, LPA produced a hyperpolarizing shift of both activation and inactivation voltages. LPA had a negligible effect on the maximal conductance of TTX-S current, but increased that of TTX-R current. The results suggest that the enhancement of TTX-R current may contribute to the LPA-induced pain.