敬钊毒素-Ⅴ的合成、复性及其钾通道活性鉴定  

Synthesis,Refolding and Characterization of JZTX-Ⅴ and Its Effect on Potassium Channels

作　　者：曾雄智[1] 邓梅春[1] 孙正华[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,长沙410081

出　　处：《中国生物化学与分子生物学报》2008年第5期463-468,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金重点项目与面上项目(No.30430170;30670640;30500146);国家重点基础研究发展规划(973计划;No.2006CB708508);国家高技术研究发展计划资助项目(863计划;No.2006AA02Z141);湖南省自然科学基金项目(No.07JJ3072;06C503)~~

摘　　要：敬钊毒素-Ⅴ(jingzhaotoxin-Ⅴ,JZTX-Ⅴ)是从敬钊缨毛蛛粗毒中纯化得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功能,应用芴甲氧羰基(Fmoc)固相方法化学合成了JZTX-Ⅴ,合成多肽经谷胱甘肽法氧化复性后,利用反相高效液相色谱(RP-HPLC)进行分离纯化.复性产物的相对分子质量经质谱测定为3605.51,而与天然毒素混合进行HPLC共洗脱实验时也只得到单一峰.全细胞膜片钳实验显示,JZTX-Ⅴ能够抑制大鼠背根神经节细胞上的A型钾电流,却对外向延迟整流钾电流没有影响,对处于静息关闭状态的A-型钾通道也表现出较高的亲和性.JZTX-Ⅴ对A型钾通道的这种抑制作用具有浓度依从性,其半数有效抑制浓度(IC50)为52.3nmol/L·JZTX-Ⅴ通过引起A型钾通道的活化曲线往去极化方向漂移,和失活曲线往超极化方向漂移来改变通道的门控特征.目前的研究结果为深入开展JZTX-Ⅴ的功能研究及分子改造奠定了基础.Jingzhaotoxin-V （JZTX-V） is a novel inhibitor of tetrodotoxin-resistant sodium channels isolated from the venom of the spider Chilobrachys fingzhao. To further explore its function, JZTX-V was synthesized using a solid-phase chemical approach with Fmoc-protected amino acids. The synthetic peptides were purified by reverse-phase HPLC and then subjected to oxidative refolding under the optimal conditions. The molecular mass of the refolded JZTX-V was determined to be 3 605.51 by MALDI-TOF/TOF MS. A unique peak was observed in the RP-HPLC chromatogram for refold JZTX-V ; and the ratio to native JZTX-V was 1 ： 1 from the mixed samples. From the whole-cell patch-clamp analyses of dorsal root ganglion neurons of adult rats, JZTX- V was able to block A-type potassium channels, but had no effect on outward delay-rectified channels. JZTX- V exhibited a high affinity to the channels in resting closed states, and its inhibition of A-type potassium currents was concentration-dependent with the ICso value of 52.3 nmol/L. JZTX-V could also alter the gating properties of A-type potassium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. The results would contribute to the deeper understanding and the molecular reconstruction of JZTX-V .

关 键 词：敬钊毒素-Ⅴ(JZTX-Ⅴ) 固相多肽合成 氧化复性 全细胞记录 钾电流 

分 类 号：Q25[生物学—细胞生物学] Q343