Lys4突变对敬钊缨毛蛛毒素-V钠通道活性的影响  

Effects of Lys4 Mutation on Sodium Channels Activity of JZTX-V

作　　者：曾雄智[1] 邓梅春[1] 彭莹[1] 魏文廷[1] 王贤纯[1] 梁宋平[1] 

机构地区：[1]湖南师范大学蛋白质化学与发育生物学教育部重点实验室,中国湖南长沙410081

出　　处：《生命科学研究》2008年第1期20-23,共4页Life Science Research

基　　金：国家自然科学基金重点项目(30430170);面上项目(30670640;30500146);国家973项目(2006CB708508);863项目(2006AA02Z141);湖南省自然科学基金资助项目(07JJ3072;06C503)

摘　　要：敬钊缨毛蛛毒素-V(jingzhaotoxin-V,JZTX-V)是从敬钊缨毛蛛粗毒中纯化到的一种新型河豚毒素不敏感型钠通道抑制剂,为了深入研究该毒素的结构与功能关系,应用芴甲氧羰基(Fmoc)固相多肽化学合成方法合成了用丙氨酸(Ala)替代JZTX-V第4位赖氨酸残基的突变体K4A-JZTX-V,合成线性多肽经反相高效液相色谱分离纯化后进行谷胱甘肽氧化复性.复性产物分别用MALDI-TOF/TOF质谱进行相对分子质量的鉴定,用膜片钳电生理方法进行电压门控钠通道抑制活性分析.研究结果表明,Lys4被Ala取代后,K4A-JZTX-V对大鼠背根神经节细胞膜上表达的河豚毒素敏感型(TTX-S)钠通道的抑制活性与天然JZTX-V基本相当,提示Lys4与JZTX-V对TTX-S钠通道的抑制活性关系不大;而K4A-JZTX-V对河豚毒素不敏感型(TTX-R)钠通道的抑制活性却比天然JZTX-V下降了约8.3倍,说明Lys4是JZTX-V与河豚毒素不敏感型钠通道抑制活性相关的氨基酸残基.Jingzhaotoxin-V （JZTX-V） isolated from the venom of the spider Chilobrachys jingrhao is a novel potent inhibitor that acts on tetrodotoxin-resistant sodium channels in adult rat dorsal root ganglion （DRG） neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structurefunction relationship of the toxin, a mutant of JZTX-V in which Lys4 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 and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of K4A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V,suggesting that Lys4 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast,the prohibitive level of K4A-JZTX-V on TTX-R sodium channels is reduced by 8.3 times,indicating that Lys4 is involved in the binding activities of JZTX-V to TTX-R sodium channels.

关 键 词：敬钊缨毛蛛毒素-V 固相多肽合成 突变体 氧化复性 膜片钳 

分 类 号：Q25[生物学—细胞生物学] Q343