蜘蛛钠离子通道抑制神经毒素的结构与功能研究

【作者】 肖玉成；

【导师】 梁宋平； 谢锦云； 陈湘定；

【作者基本信息】 湖南师范大学 ， 生物化学， 2002， 硕士

【摘要】 虎纹捕鸟蛛（Selenocosmia huwena）、海南捕鸟蛛（Selenocosmia hainana）和雷氏大疣蛛（Macrothele raveni）均是在我国近年来发现的蜘蛛新种，前两者属于捕鸟蛛科，而后者属于异仿蛛科。三种蜘蛛均能捕食昆虫和小型脊椎动物。本文利用小鼠神经细胞瘤×大鼠神经胶质细胞的杂交细胞NG108-15，通过全细胞记录（whole-cell recording）模式的膜片钳技术，检验了虎纹捕鸟蛛、海南捕鸟蛛和雷氏大疣蛛的粗毒对NG108-15细胞膜上电压门控河豚毒敏感型（TTX-S）钠电流和延迟整流钾电流的作用。结果表明，三种蜘蛛粗毒对外向延迟整流钾电流没有明显作用，但对TTX-S的快钠电流表现出较强的抑制效应。抑制效应呈量效关系。三种粗毒抑制钠电流的有效半抑制浓度(IC₅₀)相应约为：3.4mg/L；1.8mg/L和11.0mg/L。海南捕鸟蛛毒素-Ⅳ（HNTX-Ⅳ）和海南捕鸟蛛毒素-Ⅴ（HNTX-Ⅴ）是从海南捕鸟蛛粗毒中经阳离子交换和反相HPLC柱层析分离出来的两种神经毒素，MALDI-TOF质谱鉴定前者相对分子质量为3，989Da，含有三对二硫键，由35个氨基酸残基组成；后者相对分子质量为3，972Da，与前者只相差16个质子，也含有三对二硫键。HNTX-Ⅴ经491测序仪测得其氨基酸组成为：NH2-ECLGFGKGCNPSDQCCKSANLVCSRKHRWCKYEI-COOH，含35个氨基酸残基，6个半胱氨酸全部参与了二硫键的形成，该毒素对小鼠膈神经-膈肌标本有阻遏作用。与HNTX-Ⅳ的一级结构相比较，两种海南捕鸟蛛毒素只有一个残基的不同，HNTX-Ⅴ为Ala20而HNTX-Ⅳ为Ser20，故两者应为一天然突变体。在全细胞电压钳记录模式下，我们进一步观察了两毒素对SD大鼠背根神经节（DRG）细胞电压门控激活钠电流(I_Na)的影响。实验结果表明：HNTX-Ⅳ和HNTX-Ⅴ均不影响河豚毒不敏感型（TTX-R）钠电流，但对TTX-S钠电流却有明显的抑制作用，抑制作用具有浓度依从性，其IC₅₀分别为34.8nmol/L和42.3nmol/L。50nmol/L HNTX-Ⅳ作用于DRG 摘要.细胞5分钟后，TTx一s钠电流的激活与失活相以及通道稳态激活特征均无明显改变，但TTX一s钠通道能引起钠通道的半稳态失活电压有约10.lmV的超极化漂移。100二。1/L IINTX一V也不影响TTX一s钠电流的激活相和失活相，对钠通道的激活闽值和最大激活电压也无明显改变，但能引起钠通道的半稳态失活电压有7 .7mV的超极化漂移。从上可知两肤类毒素在性质上差异不大，因此我们也可以肯定肤链第20位氨基酸残基应该不是影响TTX一S钠通道的关键残基。同时两种海南捕鸟蛛毒素从影响TTX一S钠通道的方式上来看，与其它已知肤类蜘蛛毒素如6一ACTXs等通过作用于位点3引起延缓钠通道的失活时间存在明显不同，我们推测HNTX一IV和IINTx一v可能是以一种新的不同于其它蜘蛛毒素的方式来影响钠通道的，而TTX一S钠通道位点1则很有可能便是它们的作用位点，与TTX和林一eonotoxins相似。更多还原

【Abstract】 Selenocosmia huwena, Selenocosmia hainana and Macrothele raveni were identified recently as three new species of spider in China. Among them, Selenocosmia huwena and Selenocosmia hainana belong to Theraphosidae while Macrothele raveni belongs to Hexathelidae. All of them could kill insects and some small vertebrate with their venoms. In this article, using whole-cell patch-clamp technology, we observed the effects of the venoms from the three kinds of spider on the voltage-activated tetrodotoxin-sensitive (TTX-S) sodium current and delay-rectified potassium current of undifferentiated NG108-15 cells. The results showed that they had no evident effect of outword delay-rectified potassium current, while they could inhibit TTX-S fast sodium current. The inhibitions were dose-dependent. The IC50 values of them were approximate 3.4 mg/L (Selenocosmia huwena); 1.8 mg/L (Selenocosmia hainana); 11.0 mg/L (Macrothele raveni), respectively. Both Hainantoxin-IV (HNTX-IV) and. Hainantoxin-V(HNTX-V) were peptide toxins isolated from the venom of the spider Seleconosmia hainana by means of ion-exchange chromatograph and reverse phase HPLC. HNTX-IV, 3989 Da, contained 35 amino acid residues with three disulfide bonds, while the molecular weight of HNTX-V was 3972 Da and had three disulfide bonds, too. The sequence of HNTX-V had been determined by 491 sequencer: NH2-ECLGFGKGCNPSDQCCKSANLVCSRKHRWCKYEI-COOH, in which there were 35 amino acid residues and six Cys. The toxin could block neuromuscular transmission in an isolated mouse phrenic nervediaphragm preparation. HNTX-V is a natural mutant of HNTX-IV, for there is only one different residue (Ala20Ser). Under the whole-cell patch-clamp mode, we further observed the modulation of sodium channels on adult rat dorsal root ganglion (DRG) neurons by HNTX-IV and HNTX-V. Both of them had no significant effect on tetrodotoxin-resistant (TTX-R) sodium currents, but they inhibited mammal neural TTX-S sodium currents evidently. The inhibition of TTX-S sodium currents by HNTX-IV and HNTX-V was concentration-dependent with the IC50 values of 34. 8 and 42. 3 nmol/L, respectively. After 50 nmol/L HNTX-IV treatment for 5 min, the peptide didn’ t effect the active and inactive time course of sodium currents and the steady-state activation of sodium channels, but it caused a 10.1 mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation on DRG neurons. Similar to HNTX-IV, 100 nmol/L HNTX-V didn’t effect the active and inactive kinetics of currents and didn’t have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However, 100 nmol/L HNTX-V caused a 7.7 mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation. So the results indicate that the two mutants have no significant difference in the toxicity of inhibiting mammal neuron sodium channels, and Ala20 or Ser20 was not the key residue. The two peptide neurontoxins inhibite mammalian voltage-gated sodium channels through a novel mechanism distinct from other known spider toxins such as 5 -ACTXs, -agatoxins I-VI which banded receptor site 3 to slow the inactive time course of sodium channels. We suggest that HNTX-V maybe target the site 1 of sodium channels in a similar manner to -conotoxinsand TTX.更多还原