Effects of Arg20 mutation on sodium channels activity of JZTX-V.
- Author:
Xiongzhi ZENG
1
;
Meichun DENG
;
Jianhui PI
;
Miaohua QUAN
;
Xianchun WANG
;
Songping LIANG
Author Information
1. The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Substitution;
Animals;
Arginine;
genetics;
Ganglia, Spinal;
drug effects;
Mutagenesis, Site-Directed;
Mutant Proteins;
pharmacology;
Neurons;
drug effects;
Patch-Clamp Techniques;
Peptides;
chemistry;
genetics;
pharmacology;
Rats;
Sodium Channel Blockers;
pharmacology;
Sodium Channels;
drug effects;
Spider Venoms;
chemistry;
genetics;
isolation & purification;
pharmacology;
Spiders;
Tetrodotoxin;
pharmacology
- From:
Chinese Journal of Biotechnology
2008;24(7):1228-1232
- CountryChina
- Language:Chinese
-
Abstract:
Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.
