[Effects of Arg20 mutation on sodium channels activity of JZTX-V].
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.
The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China.
Xiongzhi
X
Zeng
Meichun
M
Deng
Jianhui
J
Pi
Miaohua
M
Quan
Xianchun
X
Wang
Songping
S
Liang
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics*; D005727:Ganglia, Spinal / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D051381:Rats; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013112:Spiders; D013779:Tetrodotoxin / Q000494:pharmacology
D050505:Mutant Proteins; D010455:Peptides; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013111:Spider Venoms; C518577:jingzhaotoxin-V, Chilobrachys jingzhao; D013779:Tetrodotoxin; D001120:Arginine