Thr28突变对虎纹捕鸟蛛毒素-Ⅳ钠通道活性的影响  

Effects of Thr28 Mutation on Sodium Channels Activity of HWTX-Ⅳ

作　　者：邓梅春[1] 罗旋[2] 陈汉春[1] 王军[1] 梁宋平[2] 

机构地区：[1]中南大学生命科学学院生物化学系,中国湖南长沙410013 [2]湖南师范大学生命科学学院 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》2013年第2期111-115,共5页Life Science Research

基　　金：国家重点基础研究发展计划项目(973计划)(2010CB529801);国家自然科学基金资助项目(31100764);中国博士后科学基金资助项目(2012M521536);湖南省博士后科研资助专项计划项目(2012RS4015);中南大学自由探索计划项目(2011QNZT131);中南大学博士后科学基金资助项目

摘　　要：虎纹捕鸟蛛毒素-Ⅳ(HWTX-Ⅳ)是从虎纹捕鸟蛛粗毒中分离纯化到的一种新型多肽类神经毒素,能明显抑制表达于大鼠背根神经节细胞的河豚毒素敏感型(TTX-S)钠通道.为了更好地研究该毒素的结构与功能之间的关系,采用芴甲氧羰基(Fmoc)固相多肽化学合成法合成了用谷氨酸(Glu)替代HWTX-Ⅳ第28位苏氨酸残基的突变体T28D-HWTX-Ⅳ,线性多肽合成产物经反相高效液相色谱(HPLC)分离纯化后进行谷胱甘肽氧化复性.复性产物采用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)技术鉴定分子质量,通过全细胞膜片钳电生理技术测定其电压门控钠通道药理学活性.当第28位Thr残基被Glu取代后,突变体T28D-HWTX-Ⅳ对表达于大鼠DRG细胞膜上的TTX-S钠通道的IC50值约为362 nmol/L,对TTX-S钠通道的抑制活性比天然HWTX-Ⅳ(IC50值=30 nmol/L)下降了约12倍,显示第28位的Thr残基是HWTX-Ⅳ与TTX-S型钠通道相互作用的关键活性残基.目前的研究为进一步探索HWTX-Ⅳ的结构与功能关系及新型镇痛药物的研发奠定了基础.Huwentoxin-IV （HWTX-IV）, a novel neurotoxic polypeptide toxin characterized from Chinese tarantula Ornithoctonus huwena venom, which is found to inhibit tetrodotoxin-sensitive （TFX-S） sodium channels expressed in rat dorsal root ganglion （DRG） neurons. To investigate the structure-function relationship of the toxin, a mutant form of the HWTX-IV with Thr28 substituted by Glu was synthesized by solid-phase chemistry method with Fmoc-protected amino acids. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography （RP-HPLC） and refolded with Glutathione oxidation. The relative molecular weight of the refolded product was analyzed by matrix-assisted laser resorption/ionization time-of-flight mass spectrometry. The prohibitive activity of sodium channels was analyzed by patchclamp electrophysiological experiment. Under the whole-cell patch-clamp configuration, the mutant T28D- HWTX-IV could inhibit the currents of the TTX-S sodium channel expressed on the cell membrane of rat dorsal root ganglion （DRG） neurons with IC50 value of 362 nmol/L. However, the prohibitive level of T28D-HWTX-IV on TFX-S sodium channels was reduced amino acid residue related to the interaction between by about 12 times, indicating that Thr28 was a key HWTX-IV and TI＇X-S sodium channels. The findings would facilitate further investigation of the structure-function relationship of HWTX-IV and developing drug candidate targeting TI＇X-S sodium channel in mammalian neurons.

关 键 词：虎纹毒素-IV(HWTX—IV) 突变体 固相多肽合成 氧化复性 钠通道 

分 类 号：Q518.2[生物学—生物化学]