Spider Sodium Channel Inhibition The Structure And Function Of The Neurotoxin

Selenocosmia huwena, Selenocosmia hainana and Macrothele raveni were identified recently as three new species of spider in China. Among them, Selenocosmia huwena and Selenocosmia hainana belong to Theraphosidae while Macrothele raveni belongs to Hexathelidae. All of them could kill insects and some small vertebrate with their venoms. In this article, using whole-cell patch-clamp technology, we observed the effects of the venoms from the three kinds of spider on the voltage-activated tetrodotoxin-sensitive (TTX-S) sodium current and delay-rectified potassium current of undifferentiated NG108-15 cells. The results showed that they had no evident effect of outword delay-rectified potassium current, while they could inhibit TTX-S fast sodium current. The inhibitions were dose-dependent. The IC50 values of them were approximate 3.4 mg/L (Selenocosmia huwena); 1.8 mg/L (Selenocosmia hainana); 11.0 mg/L (Macrothele raveni), respectively. Both Hainantoxin-IV (HNTX-IV) and. Hainantoxin-V(HNTX-V) were peptide toxins isolated from the venom of the spider Seleconosmia hainana by means of ion-exchange chromatograph and reverse phase HPLC. HNTX-IV, 3989 Da, contained 35 amino acid residues with three disulfide bonds, while the molecular weight of HNTX-V was 3972 Da and had three disulfide bonds, too. The sequence of HNTX-V had been determined by 491 sequencer: NH2-ECLGFGKGCNPSDQCCKSANLVCSRKHRWCKYEI-COOH, in which there were 35 amino acid residues and six Cys. The toxin could block neuromuscular transmission in an isolated mouse phrenic nervediaphragm preparation. HNTX-V is a natural mutant of HNTX-IV, for there is only one different residue (Ala20Ser). Under the whole-cell patch-clamp mode, we further observed the modulation of sodium channels on adult rat dorsal root ganglion (DRG) neurons by HNTX-IV and HNTX-V. Both of them had no significant effect on tetrodotoxin-resistant (TTX-R) sodium currents, but they inhibited mammal neural TTX-S sodium currents evidently. The inhibition of TTX-S sodium currents by HNTX-IV and HNTX-V was concentration-dependent with the IC50 values of 34. 8 and 42. 3 nmol/L, respectively. After 50 nmol/L HNTX-IV treatment for 5 min, the peptide didn' t effect the active and inactive time course of sodium currents and the steady-state activation of sodium channels, but it caused a 10.1 mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation on DRG neurons. Similar to HNTX-IV, 100 nmol/L HNTX-V didn't effect the active and inactive kinetics of currents and didn't have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However, 100 nmol/L HNTX-V caused a 7.7 mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation. So the results indicate that the two mutants have no significant difference in the toxicity of inhibiting mammal neuron sodium channels, and Ala20 or Ser20 was not the key residue. The two peptide neurontoxins inhibite mammalian voltage-gated sodium channels through a novel mechanism distinct from other known spider toxins such as 5 -ACTXs, -agatoxins I-VI which banded receptor site 3 to slow the inactive time course of sodium channels. We suggest that HNTX-V maybe target the site 1 of sodium channels in a similar manner to -conotoxinsand TTX.