Characterization of the excitatory mechanism induced by Jingzhaotoxin-I inhibiting sodium channel inactivation.

We have recently isolated a peptide neurotoxin, Jingzhaotoxin-I (JZTX-I), from Chinese tarantula Chilobrachys jingzhao venom that preferentially inhibits cardiac sodium channel inactivation and may define a new subclass of spider sodium channel toxins. In this study, we found that in contrast to other spider sodium channel toxins acting presynaptically rather than postsynaptically, JZTX-I augmented frog end-plate potential amplitudes and caused an increase in both nerve mediated and unmediated muscle twitches. Although JZTX-I does not negatively shift sodium channel activation threshold, an evident increase in muscle fasciculation was detected. In adult rat dorsal root ganglion neurons JZTX-I (1 microM) induced a significant sustained tetrodotoxin-sensitive (TTX-S) current that did not decay completely during 500 ms and was inhibited by 0.1 microM TTX or depolarization due to voltage-dependent acceleration of toxin dissociation. Moreover, JZTX-I decreased closed-state inactivation and increased the rate of recovery of sodium channels, which led to an augmentation in TTX-S ramp currents and decreasing the amount of inactivation in a use-dependant manner. Together, these data suggest that JZTX-I acted both presynaptically and postsynaptically and facilitated the neurotransmitter release by biasing the activities of sodium channels towards open state. These actions are similar to those of scorpion alpha-toxin Lqh II.     

Synthesis and characterization of huwentoxin-IV, a neurotoxin inhibiting central neuronal sodium channels.

Our previous work demonstrated that huwentoxin-IV was an inhibitor cystine knot peptide from Chinese tarantula Ornithoctonus huwena venom that blocked tetrodotoxin-sensitive voltage-gated sodium channels from mammalian sensory neurons [Peng, K., Shu, Q., Liu, Z., Liang, S., 2002. Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J. Biol. Chem. 277(49), 47564-47571]. However, the actions of the neurotoxin on central neuronal sodium channels remain unknown. In this study, we chemically synthesized native huwentoxin-IV and found that sodium channel isoforms from rat hippocampus neurons were also sensitive to native and synthetic toxins, but the toxin-binding affinity (IC(50) approximately 0.4 microM) was 12-fold lower than to peripheral isoforms. The blockade by huwentoxin-IV could be reversed by strong depolarization due to the dissociation of toxin-channel complex as observed for receptor site 3 toxins. Moreover, small unilamellar vesicle-binding assays showed that in contrast to ProTx-II from the tarantula Thrixopelma pruriens, huwentoxin-IV almost lacked the ability to partition into the negatively charged and neutral phospholipid bilayer of artificial membranes. These findings indicated that huwentoxin-IV was a sodium channel antagonist preferentially targeting peripheral isoforms via a mechanism quite different from ProTx-II.     

布比卡因对大鼠背根神经节钠电流的影响

目的 观察布比卡因对大鼠背根神经节神经元钠电流的影响，探讨椎管内麻醉的作用机制。方法 以全细胞膜片钳技术记录临床浓度的布比卡因0～1000μmol/L对急性分离大鼠背根神经元TTX-S和TTX-R钠电流的影响。钳制电压分别为-100mv和-90mv，刺激电压分别为-10mv和0mv。结果 临床浓度的布比卡因对TTX-S和TTX-R钠电流均有明显的抑制作用，布比卡因的浓度与其抑制强度呈正相关；布比卡因对TTX-S钠电流抑制的半数有效量IC50为17．069μmol/L(Hill系数为0．8988)，而TTX-R钠电流的半数有效量IC50为34．511μmol/L(Hill系数为0．9003)。结论 临床浓度的布比卡因对背根神经节神经元TTX-S和TTX-R钠电流具有抑制作用，对TTX-S钠电流的阻滞比对TTX-R钠电流的阻滞强1倍左右，其抑制程度随局麻药浓度的增加而增加。     

Two novel sodium channel inhibitors from Heriaeus melloteei spider venom differentially interacting with mammalian channel's isoforms

Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and mu-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.     

Reversal of neuropathic pain by alpha-hydroxyphenylamide: a novel sodium channel antagonist

Sodium (Na) channel blockers are known to possess antihyperalgesic properties. We have designed and synthesized a novel Na channel antagonist, alpha-hydroxyphenylamide, and determined its ability to inhibit both TTX-sensitive (TTX-s) and TTX-resistant (TTX-r) Na currents from small dorsal root ganglion (DRG) neurons. alpha-Hydroxyphenylamide tonically inhibited both TTX-s and TTX-r Na currents yielding an IC(50) of 8.2+/-2.2 microM (n=7) and 28.9+/-1.8 microM (n=8), respectively. In comparison, phenytoin was less potent inhibiting TTX-s and TTX-r currents by 26.2+/-4.0% (n=8) and 25.5+/-2.0%, respectively, at 100 microM. alpha-Hydroxyphenylamide (10 microM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block. In the chronic constriction injury (CCI) rat model of neuropathic pain, intraperitoneal administration of alpha-hydroxyphenylamide attenuated the hyperalgesia by 53% at 100mg/kg, without affecting motor coordination in the Rotorod test. By contrast, the reduction in pain behavior produced by phenytoin (73%; 100mg/kg) was associated with significant motor impairment. In summary, we report that alpha-hydroxyphenylamide, a sodium channel antagonist, exhibits antihyperalgesic properties in a rat model of neuropathic pain, with favorable sedative and ataxic side effects compared with phenytoin.     

Purification and characterization of huwentoxin-II,a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena

Abstract: A neurotoxic peptide, huwentoxin-II (HWTX-II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED₅₀ of 127 ± 54 µg/g. HWTX-II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin-I. The complete amino sequence of HWTX-II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/GIn) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX-II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.     

Effects of a toxic fraction,PhTx2, from the spider Phoneutria nikriventer on the sodium current

Summary The toxic fraction PhTx₂ of the spider Phoneutria nigriventer was studied with a modified loose patch clamp technique on frog skeletal muscle. At saturating concentration (8 g/ml) potassium currents were unaffected whereas there was a 7-fold increase in the time constant of sodium current inactivation (at –13 mV test potential). The time course of tail current deactivation was at least 3-fold slower than the control. The steady state (100 ms) inactivation and the conductance activation were shifted toward more negative potentials by 12.2 and 7.0 mV, respectively. The reversal of the sodium current was shifted 7.6 mV to more negative potential. We conclude that PhTx₂ prolongs the inactivation and deactivation processes of sodium ion channels. These effects may account for the toxicity of PhTx₂.Recipient of a Doctoral Fellowship from the Universidade Federal de AlagoasCorrespondence to P. S. L. Beirão at the above address     

The effect of dibutyryl cAMP on tetrodotoxin-sensitive and -resistant voltage-gated sodium currents in rat dorsal root ganglion neurons and the consequences for their sensitivity to lidocaine

Tetrodotoxin-sensitive (TTXS) sodium currents in dorsal root ganglia (DRG) neurons were enhanced by DcAMP applied acutely or by pre-treatment. Pre-treatment increased peak TTXS by 28%. This compared to the increase of tetrodotoxin-resistant sodium currents (TTXR) of 123%. In both cases the increase was associated with a hyperpolarizing shift in activation potentials. Slow inactivation was slower for both TTXR and TTXS in DcAMP treated neurons but rates of recovery from inactivation were not altered. Lidocaine blocked TTX-R with an IC(50) of 0.51+/-0.15mM (n=9) which was reduced to 0.14+/-0.05mM (n=8, P<0.05) in DcAMP treated cells. The sensitivity of TTX-S currents to lidocaine was not altered by DcAMP (control EC(50)=0.89+/-0.16mM, n=9; DcAMP EC(50)=0.73+/-0.19mM, n=6). It is concluded that TTXS currents in DRG are, like TTX-R currents, enhanced by cAMP but whilst the pharmacology of TTXR channels with respect to lidocaine is altered, that to TTXS channels is not.     

Potent analgesic effects of a putative sodium channel blocker M58373 on formalin-induced and neuropathic pain in rats

M58373, 4-[2-(4-hydroxy-4-{[N-(4-isopropoxyphenyl)-N-methylamino]methyl}piperidin-1-yl)ethyl]benzonitrile monohydrochloride, is a novel compound, which has an inhibitory activity on neurotoxin binding to the site 2 of voltage-gated sodium channels. In this study, we investigated the effects of M58373 on substance P release from sensory neurons in vitro and pain behaviors/responses in rats, compared with mexiletine. M58373 (1-10 microM) inhibited veratridine-induced release of substance P from dorsal root ganglion cells. In the formalin test, oral M58373 (0.3-10 mg/kg) reduced the time spent in nociceptive behaviors only in the late phase. In the neuropathic pain model, oral M58373 (1-10 mg/kg) attenuated mechanical allodynia and heat hyperalgesia in the nerve-injured paw without affecting normal responses in the uninjured paw. In contrast, oral mexiletine (10-100 mg/kg) had a narrow therapeutic dose range in both models because of the adverse effects on the central nervous system. These results suggest that M58373 is a favorable prototype for novel anti-neuropathic pain agents.     

Purification and characterization of raventoxin-I and raventoxin-III, two neurotoxic peptides from the venom of the spider Macrothele raveni.

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD(50) of the crude venom in mice was 2.852mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD(50) of raventoxin-I in mice is 0.772mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with delta-AcTx-Hv1 and delta-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.     

Isolation and characterization of a novel toxin from the venom of the spider Grammostola rosea that blocks sodium channels.

This communication reports the chemical and physiological characterization of a novel peptide (GrTx1) isolated from the venom of the "rosean-tarantula"Grammostola rosea. This component was one among more than 15 distinct components separated from the soluble venom by high-performance liquid chromatography (HPLC). GrTx1 has 29 amino-acid residues, compactly folded by three disulfide bridges with a molecular weight of 3697Da. Here we show that this peptide blocks Na(+) currents of neuroblastoma F-11 cells with an IC(50) of 2.8+/-0.1muM, up to a maximum of about 85% at 10muM. Moreover, the right-shift (+20.1+/-0.4mV) of the fractional voltage-dependent conductance could be also compatible with a putative "gating-modifier" mechanism. No effects were seen on common K(+) channels, such as K(v)1.1 and 1.4, using concentrations of toxin up to 10muM. Sequence analysis reveals that GrTx1 is closely related to other spider toxins reported to affect various distinct ion channel functions. A critical analysis of this study suggests the necessity to search for other potential receptor sites in order to establish the preferred specificity of these kind of peptides.     

A-887826 is a structurally novel, potent and voltage-dependent Nav1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na_v1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na_v1.8 channel blockers. A-887826 exemplifies this series and potently (IC₅₀ = 11nM) blocked recombinant human Na_v1.8 channels. A-887826 was ∼3 fold less potent to block Na_v1.2, ∼10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na⁺) currents and was >30 fold less potent to block Na_V1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na⁺) currents (IC₅₀ = 8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund’s adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100 nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by ∼4 mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na_v1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na_v1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na_v1.8 sodium channels in pathological pain states.     

Isolation and characterization of Jingzhaotoxin-V, a novel neurotoxin from the venom of the spider Chilobrachys jingzhao.

Jingzhaotoxin-V (JZTX-V), a 29-residue polypeptide, is derived from the venom of the spider Chilobrachys jingzhao. Its cDNA determined by rapid amplification of 3' and 5'-cDNA ends encoded an 83-residue precursor with a pro-region of 16 residues. JZTX-V inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons with IC50 values of 27.6 and 30.2 nM, respectively. Moreover, the toxin exhibits high affinity to the resting closed states of the channels. JZTX-V also inhibits Kv4.2 potassium currents expressed in Xenpus Laevis oocytes (IC50=604.2 nM), but has no effects on outward delay-rectified potassium channels expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of sodium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. The phospholipid membrane binding activity of JZTX-V is also verified using intrinsic tryptophan fluorescence analysis as well as acrylamide-quenching assays. Importantly, human multiple sodium channel subtypes are attractive targets for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.     

Isolation and chemical characterization of PwTx-II: a novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae).

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and 1H NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12+/-3 etag/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat).     

芍药苷对小鼠背根神经节TTX-S钠电流的影响

目的 本文通过观察芍药苷（Paeoniflorin,Pae）对小鼠背根神经节（DRG）细胞电压门控河豚毒素敏感型（TTX-S）钠电流的影响,探讨芍药苷的镇痛机制。方法 应用全细胞膜片钳技术在急性分离背根神经节细胞上记录河豚毒素敏感型钠电流。结果 芍药苷浓度依赖地抑制河豚毒素敏感型钠电流,其半抑制浓度（IC₅₀）为1 224.1 mmol·L^-1。芍药苷1 mmol·L^-1使河豚毒素敏感型钠通道的失活曲线向超极化方向转移约7.1 mV,并且延迟失活后通道的恢复,但对激活曲线没有影响。结论 芍药苷可能通过抑制河豚毒素敏感型钠通道,改变钠通道的动力学特征,从而发挥其镇痛作用。     

Interleukin-2 regulates membrane potentials and calcium channels via mu opioid receptors in rat dorsal root ganglion neurons

Our previous studies revealed that interleukin-2 (IL-2) exerted peripheral antinociception that was partially mediated by μ opioid receptors. No ionic explanations of this effect have yet been reported. The present study was designed to investigate effects of IL-2 on the physiological properties of capsaicin-sensitive small dorsal root ganglion (DRG) neurons, which are predominantly responsible for nociceptive transmission from the periphery to the spinal cord. Intracellualr recordings of DRG neurons were made in DRG/peripheral nerve preparation in vitro. IL-2 (10³ U/ml) produced membrane hyperpolarization of –9.4 ± 3.0 mV and this effect was blocked by β-FNA (5 μM), a μ opioid receptor antagonist. Under whole-cell patch clamp recordings, transient high-threshold Ca²⁺ currents were inhibited by –56.6 ± 11.3% by IL-2. Simultaneous calcium imaging showed that this cytokine also inhibited depolarization-evoked increase in intracellular calcium concentration. All the effects of IL-2 were blocked by naloxone (1 μM). Consistent with previous studies, DAMGO, a selective μ opioid agonist, exerted similar inhibitory effects on membrane potentials and Ca²⁺ currents.

The present results indicated that μ opioid receptors were involved in the regulatory effects of IL-2 on membrane potentials and calcium channels in DRG neurons, which may contribute to IL-2-induced peripheral analgesia.     

The presynaptic activity of huwentoxin-I, a neurotoxin from the venom of the chinese bird spider Selenocosmia huwena.

Three different types of isolated nerve-synapse preparations, guinea pig ileum, rat vas deferens and toad heart, were used to investigate the physiological activity of Huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena. The twitch response of isolated guinea pig ileum induced by electrical stimulus can be inhibited by HWTX-I. After blockage, contraction of the ileum can be induced by exogenously applied acetylcholine. HWTX-I caused the inhibition of the twitch response to electrical nerve stimulation in the rat vas deferens. After the twitch was completely inhibited, noradrenaline triggered rhythmic contraction of the vas deferens. The inhibitory effect on heart of toad induced by stimulating sympathetic-vagus nerve can be reversed by HWTX-I, although exogenously applied acetylcholine still acts as an effective inhibitor. All of these results support the conclusion that HWTX-I has the presynaptic activity that effects the release of neurotransmitter from the nerve endings of both the cholinergic synapse and the adrenergic synapse.     

An overview of peptide toxins from the venom of the Chinese bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)].

The bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)] is one of the most venomous spiders in China. The venom of this spider contains a mixture of compounds with different types of biological activity. About 400 proteins and peptides from the venom can be separated and detected by 2D electrophoresis. Of these, 14 peptide toxins have been purified and characterized from the venom of this spider, with several peptide toxins exhibiting structural similarity but high functional diversity. Most of these huwentoxins (HWTX) contain 30-40 amino acids with three disulfide bonds and adopt an "inhibitor cystine-knot" (ICK) motif in their three dimensional structure, except for huwentoxin-II (HWTX-II) which adopts a novel scaffold different from the ICK motif. As a group, the toxins possess quite different biological activities including inhibition of voltage-gated calcium and sodium channels, insecticidal activity, lectin-like agglutination, and inhibition of trypsin. Eight cDNAs encoding seven toxins, HWTX-I, -II, -III, -IIIa, -IV -V, and, -VII and one lectin, S. huwena lectin-I (SHL-I), have been cloned and sequenced. Comparison of the cDNA sequences of the eight peptides from S. huwena indicates that they can be classified into two different superfamilies according to the "prepro" region of their cDNA sequences.     

和厚朴酚对小鼠背根神经节细胞河豚毒素不敏感钠电流的影响

目的 通过观察和厚朴酚（honokiol, Hon）对河豚毒素不敏感（TTX-R）钠电流的作用,探讨它可能的镇痛机制。方法 应用酶解法急性分离小鼠背根神经节细胞,全细胞膜片钳技术记录河豚毒素不敏感钠电流。结果 和厚朴酚对河豚毒素不敏感钠电流的抑制呈现浓度依赖性,半抑制浓度（IC₅₀）为28.1 μmol·L^-1。和厚朴酚（30 μmol·L^-1）使河豚毒素不敏感钠电流密度下降48.1%,稳态激活和失活曲线分别向右和左偏移约7 mV和11.1 mV,但不影响通道的恢复时间。结论 和厚朴酚明显抑制河豚毒素不敏感钠电流,其作用可能与它的镇痛机制有关。     

JZTX-IV, a unique acidic sodium channel toxin isolated from the spider Chilobrachys jingzhao

Neurotoxins are important tools to explore the structure and function relationship of different ion channels. From the venom of Chinese spider Chilobrachys jingzhao, a novel toxin, Jingzhaotoxin-IV (JZTX-IV), is isolated and characterized. It consists of 34 amino acid residues including six acidic residues clustered with negative charge (pI=4.29). The full-length cDNA of JZTX-IV encodes an 86-amino acid precursor containing a signal peptide of 21 residues, a mature peptide of 34 residues and an intervening sequence of 29 residues with terminal Lys-Gly as the signal of amidation. Under whole-cell patch clamp conditions, JZTX-IV inhibits current and slows the inactivation of sodium channels by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. In addition, JZTX-IV shows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons, differs from the classic site 3 toxins that do not affect the steady-state of inactivation. At high concentration, JZTX-IV has no significant effect on tetrodotoxin-resistant (TTX-R) sodium channels on rat DRG neurons and tetrodotoxin-sensitive (TTX-S) sodium channels on hippocampal neurons. Our data establish that, contrary to known toxins, JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating, thus making it a novel probe of channel gating in sodium channels with potential to shed new light on this process.