Synthesis and biological characterization of synthetic analogs of Huwentoxin-IV (Mu-theraphotoxin-Hh2a), a neuronal tetrodotoxin-sensitive sodium channel inhibitor

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• 作者：Meichun Deng ; Xuan Luo ; Liping Jiang ; Hanchun Chen ; Jun Wang ; Hailun He ; Songping Liang
• 关键词：HWTX-IV ; Voltage-gated sodium channel ; Mutant ; Solid-phase peptide synthesis ; Protein folding ; Patch-clamp
• 刊名：Toxicon
• 出版年：2013
• 出版时间：1 September, 2013
• 年：2013
• 卷：71
• 期：Complete
• 页码：57-65
• 全文大小：1423 K

文摘

Huwentoxin-IV (HWTX-IV, also named Mu-theraphotoxin-Hh2a) is a typical inhibitor cystine knot peptide isolated from the venom of Chinese tarantula Ornithoctonus huwena and is found to inhibit tetrodotoxin-sensitive (TTX-S) sodium channels from mammalian sensory neurons. This peptide binds to neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II in neuronal sodium channels. However, the molecular surface of HWTX-IV interaction with sodium channels remains unknown. In this study, we synthesized HWTX-IV and three mutants (T28D, R29A and Q34D) and characterized their functions on TTX-S sodium channels from adult rat dorsal root ganglion (DRG) neurons. Analysis of liquid chromatography, mass spectrometry and circular dichroism spectrum indicated that all four synthetic peptides are properly folded. Synthetic HWTX-IV exhibited the same activity as native HWTX-IV, while three mutations reduced toxin binding affinities by 10-200 fold, indicating that the basic or vicinal polar residues Thr²⁸, Arg²⁹, and Gln³⁴ in C-terminus might play critical roles in the interaction of HWTX-IV with TTX-S sodium channels.