Hainantoxin-IV (HNTX-IV) is a peptide that was originally isolated from the venom of the Chinese bird spider Ornithoctonus hainana Liang (Selenocosmia hainana Liang). It has been reported that this peptide is a potent antagonist of tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels (VGSCs). Hainantoxin-IV binds to TTX-S with an IC₅₀value of 34 nM in adult rat dorsal root ganglion (DRG) neurons. Tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels are not affected by Hainantoxin-IV. It probably interacts with the site 1 through a mechanism quite similar to that of TTX without affecting the activation and inactivation kinetics.

Description:

Product code: N/A.Category: Sodium channels.Tags: nav, tetrodotoxin, ttx.

AA sequence: Glu-Cys²-Leu-Gly-Phe-Gly-Lys-Gly-Cys⁹-Asn-Pro-Ser-Asn-Asp-Gln-Cys¹⁶-Cys¹⁷-Lys-Ser-Ser-Asn-Leu-Val-Cys²⁴-Ser-Arg-Lys-His-Arg-Trp-Cys³¹-Lys-Tyr-Glu-Ile-NH2(Disulfide bonds between Cys²-Cys¹⁷, Cys⁹-Cys²⁴, and Cys¹⁶-Cys³¹)Length (aa): 35Formula: C₁₆₆H₂₅₇N₅₃O₅₀S₆Molecular Weight: 3987.6 DaAppearance: White lyophilized solidSolubility: water and saline bufferCAS number: Not availableSource: SyntheticPurity rate: > 97%

Reference:

A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels

Hainantoxin-IV (HNTX-IV), isolated from the venom of the spider Ornithoctonus hainana, is a specific antagonist of tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels in rat dorsal root ganglion (DRG) cells. It adopts an inhibitor cystine knot motif, and structural analysis revealed a positively charged patch consisting of Arg26, Lys27, His28, Arg29 and Lys32 distributed on its molecular surface. Our previous study demonstrated that Lys27 and Arg29 but not Arg26 were critical residues for HNTX-IV binding to TTX-S sodium channels. In the present study, we examined the roles of His28 and Lys32 in the interaction of HNTX-IV with its target. Two mutants, HNTX-IV-H28D and HNTX-IV-K32A, were generated by solid-phase chemical synthesis and purified by reverse-phase HPLC after refolding and oxidation, yielding two compounds of high purity with monoisotopic masses of 3962.66 and 3927.70 Da, respectively, as determined by MALDI-TOF mass spectrometry. This indicated the presence of six cysteine residues forming three disulfide bonds. Moreover, circular dichroism spectroscopy analysis demonstrated that the substitution of His28 or Lys32 did not affect the overall structure of HNTX-IV. The inhibitory activity of HNTX-IV-H28D and HNTX-IV-K32A against TTX-S sodium channels in rat DRG cells was analyzed by whole-cell patch-clamp technique. The IC(50) values for the mutants were 0.57 and 5.80 μM (17-fold and 170-fold lower than the activity of the native toxin), indicating that His28 and Lys32 may be important for the inhibitory activity of HNTX-IV. Taken together, our results suggest that the positively charged patch might be the binding site for the interaction of HNTX-IV with TTX-S sodium channels. These findings might contribute to the elucidation of the structure and function relationship of HNTX-IV.

Liu Y, et al. (2012) A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels. J Pept Sci. PMID: 22927181

Structure--activity relationships of hainantoxin-IV and structure determination of active and inactive sodium channel blockers.

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The sequence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH(2). In the present study, to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. (1)H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore, in the determined structures of K27A and R29A, the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV. These results suggested that residues Ser(12), Arg(26), Lys(27), and Arg(29) were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys(27) and Arg(29) were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys(27) and Arg(29) with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys(27), Arg(29), His(28), Lys(32), Phe(5), and Trp(30) clustered on one face of HNTX-IV were responsible for ligand binding.

Li D, et al.(2004) Structure–activity relationships of hainantoxin-IV and structure determination of active and inactive sodium channel blockers. J Biol Chem. PMID: 15201273

Isolation and characterization of hainantoxin-IV, a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider Selenocosmia hainana.

A neurotoxin, named hainantoxin-IV, was purified from the venom of the spider Selenocosmia hainana. The amino acid sequence was determined by Edman degradation, revealing it to be a 35-residue polypeptide amidated at its C terminal and including three disulfide bridges: Cys2-Cys17, Cys9-Cys24, and Cys16-Cys31 assigned by partial reduction and sequence analysis. Hainantoxin-IV shares 80% sequence identity with huwentoxin-IV from the spider S. huwena, a potent antagonist that acts at site 1 on tetrodotoxin-sensitive (TTX-S) sodium channels, suggesting that hainantoxin-IV adopts an inhibitor cystine knot structural motif like huwentoin-IV. Under whole-cell voltage-clamp conditions, this toxin has no effect on tetrodotoxin-resistant voltage-gated sodium channels in adult rat dorsal root ganglion neurons, while it blocks TTX-S sodium channels in a manner similar to huwentoxin-IV, and the actions of both toxins on sodium currents are very similar to that of tetrodotoxin. Thus, they define a new family of spider toxins affecting sodium channels.

Liu Z, et al.(2003) Isolation and characterization of hainantoxin-IV, a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider Selenocosmia hainana. Cell Mol Life Sci. PMID: 12827284

Inhibition of neuronal tetrodotoxin-sensitive Na+ channels by two spider toxins: hainantoxin-III and hainantoxin-IV.

Hainantoxin-III and hainantoxin-IV, isolated from the venom of the Chinese bird spider Seleconosmia hainana, are neurotoxic peptides composed of 33-35 residues with three disulfide bonds. Using whole-cell patch-clamp technique, we investigated their action on ionic channels of adult rat dorsal root ganglion neurons. It was found that the two toxins did not affect Ca2+ channels (both high voltage activated and low voltage activated types) nor tetrodotoxin-resistant voltage-gated Na+ channels (VGSCs). However, hainantoxin-III and hainantoxin-IV strongly depressed the amplitude of tetrodotoxin-sensitive Na+ currents with IC50 values of 1.1 and 44.6 nM, respectively. Both hainantoxin-III (1 nM) and hainantoxin-IV (50 nM) caused a hyperpolarizing shift of about 10 mV in the voltage midpoint of steady-state Na+ channel inactivation, but they showed difference in the reprime kinetics of VGSCs: hainantoxin-III significantly decreased the recovery rate from inactivation at a prepulse potential of -80 mV while hainantoxin-IV did not do. It is interesting to note that similar to huwentoxin-IV, the two hainantoxins did not affect the activation and inactivation kinetics of Na+ currents and at a concentration of 1 microM they completely inhibited the slowing inactivation currents induced by BMK-I (toxin I from the scorpion Buthus martensi Karsch), a scorpion alpha-like toxin. The results indicate that hainantoxin-III and hainantoxin-IV are novel spider toxins and affect the mammal neural Na+ channels through a mechanism quite different from other spider toxins targeting the neural receptor site 3, such as delta-aractoxins and mu-agatoxins.

Xiao Y, et al. (2003) Inhibition of neuronal tetrodotoxin-sensitive Na+ channels by two spider toxins: hainantoxin-III and hainantoxin-IV. Eur J Pharmacol. PMID: 14512091

Determination of disulfide bridges of two spider toxins: Hainantoxin-III and Hainantoxin-IV

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

Wang W., et al. (2009) Determination of disulfide bridges of two spider toxins: Hainantoxin-III and Hainantoxin-IV. J Venom Anim Toxins incl Trop Dis.

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