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At least 19 recordsLinked to original sources
PubMed1996

11-Oxo-tetrodotoxin and a specifically labelled 3H-tetrodotoxin.

Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na+ channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na+ channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na(+)-channel ligands.

Animals↗
PubMed2005

The anti-nociceptive agent ralfinamide inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents in dorsal root ganglion neurons.

Tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ channels contribute to the abnormal spontaneous firing in dorsal root ganglion neurons associated with neuropathic pain. Effects of the anti-nociceptive agent ralfinamide on tetrodotoxin-resistant and tetrodotoxin-sensitive currents in rat dorsal root ganglion neurons were therefore investigated by patch clamp experiments. Ralfinamide inhibition was voltage-dependent showing highest potency towards inactivated channels. IC50 values for tonic block of half-maximal inactivated tetrodotoxin-resistant and tetrodotoxin-sensitive currents were 10 microM and 22 microM. Carbamazepine, an anticonvulsant used in the treatment of pain, showed significantly lower potency. Ralfinamide produced a hyperpolarising shift in the steady-state inactivation curves of both currents confirming the preferential interaction with inactivated channels. Additionally, ralfinamide use and frequency dependently inhibited both currents and significantly delayed repriming from inactivation. All effects were more pronounced for tetrodotoxin-resistant than tetrodotoxin-sensitive currents. The potency and mechanisms of actions of ralfinamide provide a hypothesis for the anti-nociceptive properties found in animal models.

Analgesics↗
PubMed1993

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.

Allethrins↗
PubMed1988

Multiple saxitoxin-binding sites in bullfrog muscle: tetrodotoxin-sensitive sodium channels and tetrodotoxin-insensitive sites of unknown function.

The possible presence of multiple sodium channel subtypes in bullfrog skeletal muscle was investigated in binding experiments with [3H]saxitoxin and in single-channel studies using planar lipid bilayers. Two classes of [3H]saxitoxin-binding sites were identified in membrane preparations. One class displayed a toxin specificity characteristic of voltage-dependent sodium channels: high affinity for saxitoxin (KD approximately equal to 0.5 nM), neosaxitoxin (KD approximately equal to 0.1 nM), and tetrodotoxin (KD approximately equal to 1.3 nM). A second class of membrane-associated binding sites exhibited high affinity for saxitoxin (KD approximately equal to 0.1 nM), lower affinity for neosaxitoxin (KD approximately equal to 25 nM), and complete insensitivity to tetrodotoxin at concentrations up to 32 microM. The first class corresponded to functional tetrodotoxin-sensitive sodium channels that could be incorporated and observed in planar bilayers in the presence of batrachotoxin. Similar attempts to incorporate tetrodotoxin-insensitive sodium channels from bullfrog muscle and heart membranes were unsuccessful. The unusual, tetrodotoxin-insensitive binding activity for [3H]saxitoxin was also found at nM levels in the high speed supernatant of homogenized skeletal muscle without the addition of detergents. This soluble class of sites exhibited low affinity for neosaxitoxin (KD approximately equal to 60 nM) and a very slow dissociation rate of [3H]saxitoxin (t0.5 approximately equal to 90 min), properties nearly identical to those of the tetrodotoxin-insensitive sites in membranes. The soluble saxitoxin-binding activity is also characterized by a more basic pH dependence and a complete lack of binding competition between saxitoxin and alkali cations. Bullfrog muscle appears to be a good tissue source for the purification of this soluble saxitoxin-binding protein.

Amphibian Proteins↗
PubMed1994

Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

Tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channel currents were recorded from rat dorsal root ganglion neurons. The two types of sodium channel currents could be distinguished by the effects of predepolarization, 10 nM tetrodotoxin, and the inactivation during depolarization. Single-channel conductances were calculated to be 6.3 and 3.4 pS in the tetrodotoxin-sensitive and tetrodotoxin-resistant channels, respectively.

Animals↗
PubMed1995

The tetrodotoxin-insensitive sodium current in rat dorsal root ganglia is unlikely to involve the expression of the tetrodotoxin-resistant sodium channel, SkM2.

Tetrodotoxin-insensitive (TTX-I) sodium currents have been recorded from newborn and adult rat sensory neurons, but the sodium channel gene(s) responsible for the TTX-I current are unknown. Because SkM2, one of six voltage-sensitive sodium channel genes cloned from rat, encodes the only cloned channel that is relatively resistant to tetrodotoxin, we sought to test whether the TTX-I current in rat sensory neurons is due to the SkM2 channel. We hypothesized that the TTX-I current might be generated from (1) an RNA splicing variant of SkM2, (2) post-translational modification of the SkM2 protein, or (3) interaction with alternate additional channel subunits. SkM2 mRNA expression was examined in newborn rat dorsal root ganglia (DRG) by RNase protection assay. No SkM2 expression was detected. Therefore, we conclude that the TTX-I sodium current in DRG is unlikely to result from the expression of the SkM2 gene.

Alternative Splicing↗
PubMed1996

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 degrees 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 microM 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 microM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (tau fast) and 5.73 ms (tau slow). 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 amplitude of TTX-R single-channel currents was slightly changed from -0.72 +/- 0.14 to -0.83 +/- 0.07 pA by 10 microM 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 microM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (tau fast) and 9.75 ms (tau 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.

Animals↗
PubMed1991

Protection against tetrodotoxin and saxitoxin intoxication by a cross-protective rabbit anti-tetrodotoxin antiserum.

A tetrodotoxin-formaldehyde-keyhole limpet hemocyanin conjugate was used to immunize a rabbit for the production of anti-toxin antiserum. The antiserum cross-reacted against both tetrodotoxin (TTX) and saxitoxin (STX), and in a quantitative in vitro assay was able to protect cells in a dose-dependent manner from the effects of either TTX or STX. The antiserum was also able to passively protect mice challenged in vivo with either toxin. Hybridomas producing monoclonal antibodies against toxin were obtained from the spleens of mice immunized with the same conjugate.

Animals↗
PubMed2000

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.

Animals↗
PubMed2001

Kinetics of modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels by tetramethrin and deltamethrin.

Pyrethroid insecticides may be classified into two groups: type I pyrethroids lack a cyano group in the alpha-position, whereas type II pyrethroids have a cyano group. Both types prolong the sodium channel current thereby causing hyperexcitability, yet details of modulation of current kinetics remain largely to be seen. The mechanism of pyrethroid modulation of sodium currents was studied by the whole-cell patch-clamp technique with rat dorsal root ganglion neurons. Both deltamethrin (type II) and tetramethrin (type I) acted on both tetrodotoxin-sensitive and tetrodotoxin-resistant channels in a qualitatively similar manner and some quantitative differences were derived from different kinetics. During repetitive stimulation in the presence of deltamethrin, leak current increased due to accumulation of prolonged tail currents, explaining the apparent use-dependent modification. For tetramethrin-modified channels, such accumulation was much less because of faster kinetics. Slowing of the kinetics of sodium channel activation by deltamethrin was revealed even after the fast inactivation had been removed by papain. The kinetics of deltamethrin-modified sodium channels was fitted better by the equation that contained two activation components than that with one component. Deltamethrin caused a large shift of the conductance-voltage curve in the direction of hyperpolarization. Cell-attached patch-clamp experiments revealed that deltamethrin had much smaller mobility in the cell membrane than tetramethrin. It was concluded that the apparent use dependence of deltamethrin modification of sodium channels was due primarily to the accumulation of prolonged tail currents during repetitive stimulation and that the sodium channel activation mechanism is the major target of pyrethroids.

Anesthetics, Local↗
PubMed2002

Kinetic properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channel currents in neonatal rat trigeminal ganglion neurons.

The kinetic properties of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na' channels in acutely dissociated neonatal rat trigeminal ganglion neurons were studied using whole-cell and cell-attached patch-clamp recordings. The time course of TTX-R currents was slower than that of TTX-S currents. Compared with TTX-S currents, TTX-R currents had more positive half-activation and half-inactivation voltages. TTX-R currents recovered from inactivation much faster than TTX-S currents. Cell-attached patch recordings showed that the slope conductance of single TTX-S and TTX-R channels was 14.6 pS and 7.8 pS, respectively. TTX-R channels had longer open-times and more dispersed latent-times than TTX-S channels. The convolution of the first latency distribution with the open-time distribution revealed that the slower time course of TTX-R currents is due to longer open-times and more dispersed latent-times of the TTX-R channels compared with those of the TTX-S channels. These findings suggest that TTX-R Na+ channels in trigeminal ganglion neurons have similar kinetic property to brain TTX-S Na+ channels, but not to structurally homologous cardiac Na+ channels.

Action Potentials↗
PubMed1994

Differential mechanism of action of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.

Rat dorsal root ganglion neurons are endowed with tetrodotoxin-sensitive(TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels. The pyrethroid insecticides, which are known to keep sodium channels open for a prolonged period of time, cause differential effects on the two types of sodium channels. The whole-cell patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. In TTX-S sodium channels, the slow sodium current during step depolarization was increased somewhat by tetramethrin, and a tail sodium current with a slowly rising and falling phase appeared upon repolarization. The tail current developed even after the sodium current during depolarization had subsided. In TTX-R sodium channels, the slow sodium current during step depolarization was increased markedly by tetramethrin, and upon repolarization a large instantaneous tail current was generated and decayed slowly. The steady-state sodium channel inactivation curve was shifted by tetramethrin in the hyperpolarizing direction in both TTX-S and TTX-R channels. The sodium conductance-voltage curve also was shifted by tetramethrin in the hyperpolarizing direction in both TTX-S and TTX-R channels, and the latter was affected more strongly than the former. At a concentration of 10 microM, the highest concentration tested, tetramethrin modified only 12% of the TTX-S sodium channels, whereas the modification was as high as 81% in the TTX-R. Even at 10 nM, 1.3% of TTX-R sodium channels were modified; this accounts for the high potency of tetramethrin as an insecticide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Downregulation of tetrodotoxin-resistant sodium currents and upregulation of a rapidly repriming tetrodotoxin-sensitive sodium current in small spinal sensory neurons after nerve injury.

Clinical and experimental studies have shown that spinal sensory neurons become hyperexcitable after axonal injury, and electrophysiological changes have suggested that this may be attributable to changes in sodium current expression. We have demonstrated previously that sodium channel alpha-III mRNA levels are elevated and sodium channel alpha-SNS mRNA levels are reduced in rat spinal sensory neurons after axotomy. In this study we show that small (C-type) rat spinal sensory neurons express sodium currents with dramatically different kinetics after axotomy produced by sciatic nerve ligation. Uninjured C-type neurons express both slowly inactivating tetrodotoxin-resistant (TTX-R) sodium current and a fast-inactivating tetrodotoxin-sensitive (TTX-S) current that reprimes (recovers from inactivation) slowly. After axotomy, the TTX-R current density was greatly reduced. No difference was observed in the density of TTX-S currents after axotomy, and their voltage dependence was not different from controls. However, TTX-S currents in axotomized neurons reprimed four times faster than control TTX-S currents. These data indicate that axotomy of spinal neurons is followed by downregulation of TTX-R current and by the emergence of a rapidly repriming TTX-S current and suggest that this may be attributable to the upregulation of a sodium channel isoform that was unexpressed previously in these cells. These axotomy-induced changes in sodium currents are expected to alter excitability substantially and could underlie the molecular pathogenesis of some chronic pain syndromes associated with injury to the axons of spinal sensory neurons.

Anesthetics, Local↗
PubMed1997

Differential action of riluzole on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.

The effects of riluzole, a neuroprotective drug, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. At the resting potential, riluzole preferentially blocked TTX-S sodium channels, whereas at more negative potentials, it blocked both types of sodium channels almost equally. The apparent dissociation constants for riluzole to block TTX-S and TTX-R sodium channels in their resting state were 90 and 143 microM, respectively. Riluzole shifted the voltage dependence of activation of TTX-R sodium channels in the depolarizing direction more than that of TTX-S sodium channels. The voltage dependence of the fast inactivation of both types of sodium channels was shifted in the hyperpolarizing direction in a dose-dependent manner, and the apparent dissociation constants for riluzole to block the inactivated channels were estimated to be 2 and 3 microM for the TTX-S and TTX-R sodium channels, respectively, indicating a much higher affinity for the inactivated channels than for the resting channels. Riluzole was equally effective in blocking both types of sodium channels in their slow inactivated state. Since more TTX-S channels are inactivated than TTX-R channels at the resting potential, riluzole blocks TTX-S sodium channels more potently than TTX-R sodium channels. It was concluded that one of the mechanisms by which riluzole exerts its neuroprotective action is to preferentially block the inactivated sodium channel of damaged or depolarized neurons under ischemic conditions, thereby suppressing excess stimulation of the glutamatergic receptors and massive influx of Ca++.

Animals↗
PubMed1998

Potent modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels by the type II pyrethroid deltamethrin.

In both tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium channels, deltamethrin greatly prolonged the current during step depolarizing pulse and caused a large and prolonged slow tail current. The activation was shifted by 20 mV in the hyperpolarizing direction. These changes in channel kinetics account for the prolongation of action potential, membrane depolarization and spontaneous discharges in the deltamethrin-treated neurons. The slow tail current of TTX-S sodium channels rose and decayed slowly, showing a hook. By contrast, the slow tail current of TTX-R channels occurred quickly upon step repolarization. The slow tail current in deltamethrin-treated cells developed slowly during a depolarizing pulse, with a time constant in the order of several milliseconds. The percentages of sodium channels modified by deltamethrin were measured as a function of the deltamethrin concentration. The EC50 values were 0.53 microM and 0.37 microM for TTX-S and TTX-R sodium channels, respectively. However, when compared at the level of 5% modification, the potency of deltamethrin for TTX-R channels was 40 to 50 times higher than that for TTX-S channels. Deltamethrin-induced large and prolonged tail current was hardly reversed after prolonged washout with drug-free solution. However, after application of tetramethrin, it was converted into a much shorter tail current. Washout with solution devoid of tetramethrin and deltamethrin resulted in rapid reappearance of the deltamethrin-type tail current. These results suggest that the deltamethrin and tetramethrin share a binding site on the sodium channel and that the slow onset and offset of deltamethrin action are controlled by the rates at which deltamethrin moves and unbinds from the membrane lipid phase rather than by the rate of deltamethrin binding to the sodium channel site.

Action Potentials↗
PubMed1986

Tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ channels differ in their sensitivity to Cd2+ and Zn2+.

The sensitivity of Na+ channels to inhibition by Cd2+ and Zn2+ was studied in 22Na+ uptake experiments after stabilization of an open conformation of the Na+ channels with different neurotoxins and in voltage clamp experiments. Six different cell types of neuronal, cardiac or skeletal muscle origin were surveyed. Three cell types possess Na+ channels that are highly sensitive to tetrodotoxin (TTX) (Kd = 1-5 nM) and three possess Na+ channels that are resistant to TTX (Kd = 0.3-1 microM). The 22Na+ uptake experiments using veratridine or batrachotoxin to activate Na+ channels indicated that TTX-resistant Na+ channels are more sensitive to the inhibitory action of Cd2+ (IC50(Cd2+) = 0.2 mM) and of Zn2+ (IC50(Zn2+) = 50 microM) than TTX-sensitive Na+ channels (IC50(Cd2+) = 5 mM, IC50(Zn2+) = 2 mM). Electrophysiological experiments showed that high concentrations of Cd2+ (IC50 = 2 mM) are necessary to inhibit both TTX-sensitive and TTX-insensitive Na+ channels when the channels are activated by voltage steps. The results suggest that Cd2+ acts competitively with veratridine or batrachotoxin and that the difference in the effects of Cd2+ and Zn2+ on 22Na+ fluxes in TTX-sensitive and TTX-resistant cells is related to differences at the site of action of alkaloid neurotoxins.

Animals↗
PubMed1993

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Ammonium Sulfate↗
PubMed2005

Immunologic protection of anti-tetrodotoxin vaccines against lethal activities of oral tetrodotoxin challenge in mice.

Tetrodotoxin (TTX) is a high toxic small molecular neurotoxin. Haptenic vaccine for TTX was investigated and the carrier proteins were compared. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH) and tetanus toxoid (TT) via formaldehyde to form the artificial antigen TTX-TTH and TTX-TT. BALB/c mice were immunized with the artificial antigen, the TTX-specific antibody response were detected. The immunized animals were intragastrically challenged with increasing doses of TTX repeatedly. The mice which exposed to TTX in doses of 600, 630, 800, 1200, 1500, 2000 and 2400 microg/kg survived at rates of 100, 100, 90, 90, 80, 50 and 20%, with a LD(50) value of 2020 microg/kg for TTH-TTX vaccine, and of 100%, 90.9%, 90.9%, 90.9%, 63.6%, 27.3% and 0%, with a LD(50) value of 1410 microg/kg for TT-TTX vaccine, respectively. All control mice inoculated with carrier protein TTH or TT uniformly died of a dose of 600 microg/kg TTX i.g. challenge. Animals immunized with vaccines could antagonize repeated TTX challenge, half of them surviving about 6 mg/kg, and a few being able to bear a maximal accumulative dose as high as approximate 9 mg/kg of TTX challenges within eight months. The TTH-TTX vaccine was of the more excellent in protective effect from TTX oral intoxication, mainly resulted from the higher antibody affinity than that from TT-TTX vaccine. The present study for the first time demonstrated that the anti-TTX experimental vaccines would high effectively protect animal from multiple, oral TTX intoxication. Immunoprophylaxis would be the hopeful means against TTX poisoning.

Administration, Oral↗