Structure-function Study Of Voltage-gated Sodium Channel Toxins From Spider Venoms

There are three species of Chinese spiders Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)], Selenocosmia hainana Liang [=Ornithoctonus hainana (Liang)] and Macrothele raveni distributed in the south of China. Their venoms can kill insects and even some small vertebrates. Under whole-cell patch-clamp recording, we observed the effects of these venoms on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels (VGSCs) and delay-rectified potassium channels in undifferentiated NG108-15 cells. It was found that they all had no evident effect on outward delay-rectified potassium currents, while they inhibited TTX-S sodium currents in a dose-dependent mode. Their IC50 values were approximately 3.4 (S. huwena), 1.8 (S, hainana) and 11.0 mg/L (M raveni), respectively. Interestingly, different from other known spider toxins, these spider venoms affected neither channel activation kinetics nor inactivation kinetics, suggesting that there might be some novel sodium channel toxins unreported before.A novel neurotoxic peptide, named hainantoxin-V (HNTX-V), was isolated further from the venom of the Chinese bird spider S. hainana by ion-exchange chromatography and reverse-phase high performance liquid chromatography (rp HPLC). Moreover, we characterized the actions of the novel toxin and other three hainantoxins, including hainantoxin-I (HNTX-I), hainantoxin-III (HNTX-III) and hainantoxin -IV (HNTX-IV), on ion channels expressing in either adult rat dorsal root ganglion (DRG) neurons or Xenopus laevis oocytes using both whole-cell patch-clamp and two-electrode voltage-clamp techniques. Four HNTXs are sodium channel toxins composed of 33-35 residues including six cysteines, which can be linked with three disulfide bonds (I-IV, II-V and III-VI). HNTX-V is a naturally occurring mutant of HNTX-IV for there is only one residue (Ser20Ala) different between them. NMR and homologue modeling analysis indicated that all of them are inhibitor cystine knot (ICK) peptides. Four HNTXs lost to affect tetrodotoxin-resistant(TTX-R) VGSCs in DRG neurons, but they except HNTX-I strongly inhibited the activation of TTX-S VGSCs with the IC50 values of 1.1, 44.6 and 46.8 nM, respectively. HNTX-I was sensitive to rNavl.2/pl and para/tipE, which were not expressed in DRG neurons. HNTX-I blocked the two kinds of VGSCs with IC50 values of 68.0 and 4.3 uM, respectively. Additionally, HNTX III-V caused a 10 mV hyperpolarizing shift in the voltage midpoint of steady-state inactivation. Similar to the crude venom, four HNTXs neither slowed channel inactivation nor shifted the voltage-current relationship of VGSCs. Importantly, HNTX-III and HNTX-IV at 1 uM inhibited the slowing inactivation currents induced by BMK-I (a typical scorpion a-like toxin from the scorpion Buthus martensi Karsch) completely. The results indicate that four HNTXs are novel spider toxins modifying the mammal neural VGSCs through a mechanism quite different from other spider toxins targeting neural receptor site 3, such as 5-atracotoxins and u,-agatoxins. Thus, we assumed that the HNTXs should not be site 3 toxins but belong to the family of site 1 toxins. In order to determine the key residues of HNTXs responsible for binding VGSCs, four mutants of native HNTX-IV (Serl2Ala, Arg26AIa, Lys27Ala and Arg29Ala) were synthesized by solid-phase method. When checked on DRG neurons, the inhibiting abilities for Serl2Ala and Arg26Ala were similar to that for native HNTX-IV, but the IC50 values of Lys27Ala and Arg29Ala are 100 times as big as that of native HNTX-IV. Thus, Lys27 and Arg29 should be important for HNTX to bind VGSCs.Chilobrachys jingzhao was identified recently as a new species of spider in the Hainan province of China. By means of ion-exchange and rp HPLC, we have isolated two main components, denoted jingzhaotoxin-I (JZTX-I) and jingzhaotoxin-III (JZTX-III), from its venom. The molecular weights of two toxins were determined to be 3675.6 and 3919.2 Da by MALDI-TOF mass spectrometry, respectively. JZTX-I is composed of 33 residues while JZTX-III has 36 residues.