title: Synthesis and biological characterization of synthetic analogs of Huwentoxin-IV (Mu-theraphotoxin-Hh2a), a neuronal tetrodotoxin-sensitive sodium channel inhibitor.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Life Sciences, Central South University, Changsha, Hunan 410013, China. dengmch@csu.edu.cn
authors.forename: Meichun
authors.initials: M
authors.lastname: Deng
authors.forename: Xuan
authors.initials: X
authors.lastname: Luo
authors.forename: Liping
authors.initials: L
authors.lastname: Jiang
authors.forename: Hanchun
authors.initials: H
authors.lastname: Chen
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Hailun
authors.initials: H
authors.lastname: He
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D026941:Sodium Channel Blockers / Q000138:chemical synthesis / Q000494:pharmacology*; D015222:Sodium Channels / Q000378:metabolism; D013111:Spider Venoms / Q000138:chemical synthesis* / Q000494:pharmacology; D013112:Spiders / Q000737:chemistry; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000737:chemistry*
chemical_list: D009498:Neurotoxins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013111:Spider Venoms; C470632:huwentoxin IV, Selenocosmia huwena; D013779:Tetrodotoxin
keywords: DRG; Dorsal root ganglion; HPLC; HWTX; HWTX-IV; High pressure liquid chromatography; Huwentoxin; IC(50); ICK; Inhibitor cystine knot; Kv channel; MALDI-TOF; Matrix assisted laser desorption/ionization time-of-flight; Median inhibitory concentration; Mutant; Patch-clamp; Protein folding; Solid-phase peptide synthesis; TTX; TTX-R; TTX-S; TTX-resistant; TTX-sensitive; Tetrodotoxin; VGSC; Voltage-gated potassium channel; Voltage-gated sodium channel