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A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels.
J Pept Sci. 2012 Oct; 18(10):643-9.JP

Abstract

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.

Authors+Show Affiliations

College of Life Science, Hunan Normal University, Changsha, 410081, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22927181

Citation

Liu, Yu, et al. "A Positively Charged Surface Patch Is Important for hainantoxin-IV Binding to Voltage-gated Sodium Channels." Journal of Peptide Science : an Official Publication of the European Peptide Society, vol. 18, no. 10, 2012, pp. 643-9.
Liu Y, Li D, Wu Z, et al. A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels. J Pept Sci. 2012;18(10):643-9.
Liu, Y., Li, D., Wu, Z., Li, J., Nie, D., Xiang, Y., & Liu, Z. (2012). A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels. Journal of Peptide Science : an Official Publication of the European Peptide Society, 18(10), 643-9. https://doi.org/10.1002/psc.2451
Liu Y, et al. A Positively Charged Surface Patch Is Important for hainantoxin-IV Binding to Voltage-gated Sodium Channels. J Pept Sci. 2012;18(10):643-9. PubMed PMID: 22927181.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels. AU - Liu,Yu, AU - Li,Dan, AU - Wu,Zhe, AU - Li,Jing, AU - Nie,Dongsong, AU - Xiang,Yang, AU - Liu,Zhonghua, Y1 - 2012/08/27/ PY - 2012/05/27/received PY - 2012/08/03/revised PY - 2012/08/06/accepted PY - 2012/8/29/entrez PY - 2012/8/29/pubmed PY - 2014/4/2/medline SP - 643 EP - 9 JF - Journal of peptide science : an official publication of the European Peptide Society JO - J Pept Sci VL - 18 IS - 10 N2 - 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. SN - 1099-1387 UR - https://www.unboundmedicine.com/medline/citation/22927181/A_positively_charged_surface_patch_is_important_for_hainantoxin_IV_binding_to_voltage_gated_sodium_channels_ DB - PRIME DP - Unbound Medicine ER -