Molecular Basis Of Spider Toxins Interacting With Voltage-gated Ion Channel

Jingzhaotoxin-Ⅲ(JZTX-Ⅲ) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, which can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes and cause a depolarization shift about 10 mV in activation. In this study, we showed that JZTX-Ⅲwas efficiently expressed by the secretary pathway in yeast. CD spectra of JZTX-Ⅲmutants and the expressed JZTX-Ⅲalmost overlapped completely with that of the native toxin, indicating that all expressed toxins did not significantly change the secondary structure of JZTX-Ⅲ. Like the native JZTX-Ⅲ, the expressed JZTX-Ⅲinhibited Nav1.5 channel with the IC50 value of 348.0±67.9 nM and cause a depolarization shift about+13 mV in activation. Alanine-scanning analysis indicated that two acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by four Trp residues (8,9,28 and 30), play very important roles in the binding of JZTX-III to Navl.5. JZTX-III docked to Nav1.5 DIIS3-S4 linker. Mutants S799A, R800A and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-Ⅲselectively interacting with Nav1.5. The reverse mutants D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-Ⅲ. Conversely, the mutant R800D in Nav1.5 decreased JZTX-Ⅲ's IC50 by 72-fold. Therefore, our result indicated that JZTX-Ⅲis a site 4 toxin, but do not possess the same critical residues on sodium channels with other site 4 toxins.A variety of posttranslational modifications of peptides have been found in venomous animal, mostly in cone snails. A novel posttranslational modified spider toxin MHWTX-Ⅳwas purified from Chinese tarantula Ornithoctonus huwen. Mass spectrum showed than the molecular weight of MHWTX-Ⅳwas 18 Da lower than that of the native Huwentoxin-Ⅳ. Sequence analysis by mass spectrum indicated that it was highly similar to spider toxin Huwentoxin-Ⅳexcept that the N-terminal glutamic acid is replaced by pyroglutamic residue. Like the native HWTX-Ⅳ, the modified HWTX-Ⅳ(MHWTX-Ⅳ) could inhibit trodotoxin-resistant VGSCs with an IC50 value of 54.16±7.3 nM in dorsal root ganglion neurons, but not change the activation or inactivation kinetics. However, in contrast to the dissociation of HWTX-Ⅳfrom sodium channel at extreme depolarization, the inhibition of TTX-S sodium channels by MHWTX-Ⅳcould not reversed by even stronger depolarization (+200 mV,500ms). It is possible that the loss of negative charge induced by glutaminyl cyclization makes the modified toxin binding stronger. To our knowledge, this is the first report of pyroglutamic acid residue found in spider toxin and the modification could increase the trap ability to voltage sensor of sodium channel.We have isolated and characterized a novel neurotoxin named JZTX-34 from the tarantula Chilobrachys jingzhao and successfully expressed by yeast. JZTX-34 is a C-terminally amidated peptide composed by 35 amino acid residues including six cysteine residues with three disulfide bridges. JZTX-34 selectively inhibits TTX-Ssodium channels of DRG neurons with the IC50 value of 85 nM, while having no effect on TTX-R sodium channels. JZTX-34 shows no effect on activation of TTX-S sodium channels but causing a depolarization shift about 10 mV on steady-state sodium channel inactivation. The inhibition by JZTX-Ⅲwas completely reversed after a high depolarization (+120 mV). Based on those results, we propose that JZTX-34 may be a site 4 toxin and trap the voltage sensor of sodium channel.The Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders in south China, its venom contains various peptide toxins and can quickly kill prey. Huwentoxin-Ⅰ(HWTX-Ⅰ) and huwentoxin-Ⅱ(HWTX-Ⅱ) are two abundant toxic components in the venom of O. huwena. HWTX-Ⅰinhibits both sodium channel (IC50～57.40±1.42 nM) and calcium channel (IC50～91 nM) of adult rat dorsal root ganglion (DRG) neurons. HWTX-Ⅱshows no effect on both sodium channel and calcium channel. When two toxins were mixed together, the activity of HWTX-Ⅰincreases 10 fold on sodium channel and 6 fold on calcium channel. HWTX-Ⅱnot only increases the activity of HWTX-Ⅰbut also accelerates the binding rate to sodium channel with about 2 fold, while having no effect on activation or inactivation kinetic. These results reveal synergistic interactions among insect and mammalian toxins and imply a new functional role for polypeptides toxins with weak activity.