Structure-function Study Of The Peptide Toxins From The Spider Chilobrachys Jingzhao And The Spider Ornithoctonus Huwena

Chinese tarantula Chilobrachys jingzhao is one of the largest venomous spiders spread in the south of China. We have isolated and characterized at least seven ion channel inhibitors from the venom of the tarantula species; we selected four peptide components (JZTX-V、-IX、-X、-XI) for further study. In order to determine the key residues of JZTX-V responsible for binding voltage-gated sodium channels, nineteen mutants of native JZTX-V were synthesized by solid-phase method. When checked on dorsal root ganglion (DRG) neurons, the ICso values of the 15 mutants on tetrodotoxin-resistant (TTX-R) sodium channels are 13-531 times as big as that of native JZTX-V, thus, these residues should be important for JZTX-V to bind TTX-R sodium channels; furthermore, the ICso values of the 5 mutants on tetrodotoxin-sensitive (TTX-S) sodium channels are 7-70 times as big as that of native JZTX-V, thus, these residues should be important for JZTX-V to bind TTX-S sodium channels.We have isolated and characterized a novel neurotoxin named Jingzhaotoxin-IX (JZTX-IX) from the venom of the tarantula. JZTX-IX is a C-terminally amidated peptide composed of 35 amino acid residues including six cysteine residues with three disulfide bridges. The toxin shows 82% sequence similarity with CcoTx3 from southeastern Africa tarantula Ceratogyrus cornuatus. To better understand their structure-function relationships, we constructed the model of JZTX-IX based on the NMR structure of its homologous toxin, JZTX-IX adopts an ICK motif composed of double anti-parallelβ-strands. JZTX-IX was found to interact with multiple types of ion channels including voltage-gated sodium channels (both tetrodotoxin-resistant and tetrodotoxin-sensitive isoforms) and Kv2.1 channel. JZTX-IX altered the gating properties of TTX-S、TTX-R sodium channels and Kv2.1 channels by causing a depolarization shift of about 10 mV in the threshold of the initial activated voltage or the active voltage of peak inward currents but not affected steady-state sodium channel inactivation. In addition, JZTX-IX could bias the activities of ion channels towards closed state because the time constant for decay (channel deactivation) of tail currents became faster in the presence of toxin. Moreover, the toxin exhibits high affinity to the resting closed states of the TTX-S、TTX-R sodium channels and Kv2.1 channels. We propose that JZTX-IX is a gating modifier showing low selectivity for ion channel types and trapping voltage sensor at closed state. The further study indicated that JZTX-IX preferentially inhibits neuronal VGSC subtype hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the three VGSCs examined, hNavl.7 was most sensitive to JZTX-IX (IC5o-110 nM). Site-directed mutagenesis analysis of sodium channels indicated that the mutants of His754、Phe813 and Arg830 located at the S3-S4 segment greatly decreased the sensitivity of hNav1.7 to JZTX-IX.Our data showed that the toxin docked at neurotoxin receptor site 4 located at the extracellular S1-S2 and S3-S4 linker of domain II. Site-directed mutagenesis analysis of potassium channels indicated that the mutants of Glu215、Phe274、Glu277、Gln284 and Phe285 greatly decreased the sensitivity of Kv2.1 to JZTX-IX. Among them, Phe274 and Phe285 are most important. JZTX-XI contains 34 residues including six cysteine residues with three disulfide bridges. The previous study indicate that JZTX-XI exhibits specific interaction against the Nav channels of rat cardiac myocytes with a significant reduction in the peak current(IC5o-0.48μM). Using whole-cell patch-clamp technique, we investigated its action on voltage-gated sodium channel isoforms. Whole-cell configuration experiment indicated that JZTX-XI was a novel neurotoxin preferentially inhibiting Nav 1.5 channel (IC50-1μM). JZTX-XI altered the gating properties of Nav 1.5 channels by causing a depolarization shift of about 10 mV in the threshold of the initial activated voltage or the active voltage of peak inward currents and a hyperpolarizing shift of about 8 mV in the voltage midpoint of steady-state sodium channel inactivation. In addition, JZTX-XI could bias the activities of Nav 1.5 channels towards closed state because the time constant for decay (channel deactivation) of tail currents became faster in the presence of toxin. We propose that JZTX-XI is a gating modifier of sodium channels and trapping voltage sensor at closed state.JZTX-X was a neurotoxin from the Chilobrachys jingzhao venom. JZTX-X contains 31 residues including six cysteine residues with three disulfide bridges. Using whole-cell patch-clamp technique and two-microelectrode voltage clamp technique, we investigated its actions on voltage-gated ion channels. JZTX-X could inhibit both Kv4.2 and Kv4.3 channels with IC50 values of 68 and 210 nM, respectively. The inhibition by JZTX-X was in a time-dependent manner and the inhibition by the toxin is completely reversible. Because JZTX-X shifted the voltage dependence of channel activation to more positive voltages, we propose that JZTX-X is a gating modifier of both Kv4.2 and Kv4.3 channels and trapping voltage sensor.The Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders in China, its venom can kill insects and even some small vertebrates. Huwentoxin-XVI (HWTX-XVI), a mammalian neurotoxic peptide, was purified from the venom of the tarantula. HWTX-XVI consists of 39 amino acid residues including six cysteines involved in three disulfide bridges. Under whole cell recording, HWTX-XVI was found to significantly inhibited N-type calcium channels in rat dorsal root ganglion cells (IC50-60 nM) while having no evident effect on voltage-gated potassium and sodium channels. The inhibition by HWTX-XVI was in a time-dependent manner and the blockage by the toxin is completely reversible. In addition, HWTX-XVI had obvious effect on the twitch response of rat vas deferens to low-frequency electrical stimulation. These results suggest that the molecular target of HWTX-XVI is very similar to that ofω-conotoxins GVIA and MVIIA. In our study, intraperitoneal injection of the toxin HWTX-XVI to rats elicited significant analgesic responses to formalin-induced inflammation pain. Intramuscular injection of the toxin also reduced mechanical allodynia induced by incisional injury in Von Frey test. Furthermore, the toxin also affect on changing withdrawal latency in hot plate tests. Thus, considering the significance of N-type calcium channels for pain transduction, HWTX-XVI may have therapeutic potential as a novel analgesic agent.In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive toω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of-51.3 mg/L. We propose that the substitutions D816G and E818S located at the extracellular S3-S4 linker of domain II in O. huwena Nav channels mayo greatly decreased toxin sensitivity of Nav 1.7 to HWTX-IV. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.