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.