【作者】 王美迟；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 1.敬钊缨毛蛛是2001年在我国海南省乐东县发现的捕鸟蛛科新种,结合阳离子交换和反相高效液相色谱技术,我们从粗毒中分离到两种新的多肽。对其测序并分别命名为敬钊缨毛蛛毒素-Ⅱ（JZTX-Ⅱ）和敬钊缨毛蛛毒素-Ⅳ（JZTX-Ⅳ）。分别描述如下:（a）JZTX-Ⅱ由32个氨基酸残基组成,选择性作用于大鼠心肌细胞,延缓其河豚毒素不敏感（TTX-R）钠通道的失活,IC₅₀为0.26±0.09μM。虽然对大鼠DRG神经元上TTX-R型钠通道没有影响,但对其河豚毒素敏感（TTX-S）型钠通道有延缓失活的活性并伴有微弱的抑制峰电流活性,其IC₅₀为0.83±0.15μM。JZTX-Ⅱ还有延缓昆虫神经元TTX-S钠通道失活的活性,其IC₅₀=1.9±0.07μM。对于大鼠海马神经元TTX-S钠通道,JZTX-Ⅱ没有表现有意义的影响。除了与JZTX-Ⅰ有56%的序列一致性外,JZTX-Ⅱ与所有已知的多肽毒素序列同源性都低,预示着JZTX-Ⅱ是一种新型、高选择性蜘蛛多肽毒素。（b）JZTX-Ⅳ由34个氨基酸残基组成,其C末端被酰胺化,是一个比较少见的酸性多肽。电生理活性实验表明JZTX-Ⅳ能抑制大鼠背根神经节（DRG）神经元TTX-S钠通道的失活,伴有弱的抑制峰电流的活性,其IC₅₀=1.77±0.29μM,JZTX-Ⅳ还使稳态激活曲线往去极化方向漂移,与经典的位点4毒素相反。虽然JZTX-Ⅳ对大鼠DRG神经元上的TTX-R钠通道不敏感,但有明显抑制心肌细胞TTX-R钠通道的作用,其IC₅₀=0.90±0.17μM,而且使稳态失活曲线往超极化方向漂移,不同于经典的位点3毒素。研究表明JZTX-Ⅳ对海马神经元钠通道没有表现活性。2.HWTX-Ⅰ是虎纹粗毒中含量最高的毒素,一种突触前N-型钙离子通道抑制剂。实验证明HWTX-Ⅰ对大鼠DRG神经元N-型钙通道和TTX-S钠通道具有抑制作用,半有效浓度分别为55nM和100nM。研究结果表明HWTX-Ⅰ对钙通道的选择性高于MVIIA,与GVIA有相同的专一性。同时,HWTX-Ⅰ是第一个在虎纹捕鸟蛛粗毒中被发现的类似于μ0-conotoxins MrVIA和MrVIB的跨通道活性多肽。我们从头化学合成了HWTX-Ⅰ,复性后的HWTX-Ⅰ通过质谱、反相色谱和电生理活性实验鉴定表明合成HWTX-Ⅰ具有与天然HWTX-Ⅰ完全相同的性质和功能。结果表明合成的HWTX-Ⅰ能完全抑制蜚蠊背侧不成对中间（DUM）神经元和大鼠海马神经元钠电流,半有效浓度分别为4.58±0.58nM和66.1±0.52nM。进一步研究表明高于IC₅₀的浓度下,HWTX-Ⅰ对大鼠DRG、海马、蜚蠊DUM神经元的稳态激活和稳态失活曲线均没有影响。3.海南捕鸟蛛毒液中分离到海南捕鸟蛛毒素-Ⅶ（HNTX-Ⅶ）组成,HNTX-Ⅶ由33个氨基酸残基组成。电生理学实验结果显示HNTX-Ⅶ能抑制大鼠DRG神经元的TTX-R和TTX-S钠通道,也抑制大鼠海马神经元的TTX-S钠通道和蜚蠊DUM神经元的TTX-S钠通道,其IC₅₀分别为0.676±0.049μM、0.169±0.046μM、1.10±0.09μM、15.1±2.7nM。HNTX-Ⅶ对大鼠TTX-S钠通道和TTX-R钠通道的激活曲线和失活曲线没有明显影响,但可使大鼠海马神经元TTX-S钠通道激活曲线往去极化方向漂移10mV,失活曲线往超极化方向漂移13.4mV。HNTX-Ⅶ对不同钠通道亚型动力学有不同影响,也许是一个很好的研究钠通道动力学的工具分子。研究同时表明HNTX-Ⅶ是一种强的昆虫钾通道抑制剂,其IC₅₀为13.7±1.2nM。更多还原

【Abstract】 1.Chilobrachys jingzhao was found in Ledong county,Hainan province in 2001.In this study,we isolated and characterized two novel VGSC toxin named jingzhaotoxin-Ⅱ（JZTX-Ⅱ） and Jingzhaotoxin-Ⅳ（JZTX-Ⅳ） from the tarantula Chilobrachys jingzhao venom.（a） JZTX-Ⅱconsists of 32 amino acid residues including two acidic and two basic residues.Cloned and sequenced using 3’- and 5’-rapid amplification of the cDNA ends,the full-length cDNA for JZTX-Ⅱwas found to encode a 63-residue precursor.Under whole-cell voltage-clamp conditions,JZTX-Ⅱsignifycantly slowed rapid inactivation of TTX-resistant （TTX-R） VGSC on cardiac myocytes with the IC₅₀=0.26±0.09μM.In addition,JZTX-Ⅱhad no effect on TTX-R VGSCs on rat dorsal root ganglion neurons but exerted a concentration-dependent reduction in tetrodotoxin-sensitive（TTX-S） VGSCs accompanied by a slowing of sodium current inactivation similar to delta-ACTXs.It is notable that TTX-S VGSCs on cultured rat hippocampal neurons were resistant to JZTX-Ⅱat high dose.Based on its high selectivity for mammalian VGSC subtypes,JZTX-Ⅱmight be an important ligand for discrimination of VGSC subtypes and for exploration of the distribution and modulation mechanisms of VGSCs.（b） Jingzhaotoxin-Ⅳ（JZTX-Ⅳ） was isolated and characterized.It consists of 34 amino acid residues including six acidic residues clustered with negative charge （pI=4.29）.The full-length cDNA of JZTX-Ⅳencodes an 86-amino acid precursor containing a signal peptide of 21 residues,a mature peptide of 34 residues with terminal Lys-Gly as the signal of amidation.Under whole-cell patch clamp conditions,JZTX-Ⅳinhibits current and slows the inactivation of sodium channels(IC₅₀=1.77±0.29μM) by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons,therefore,differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction.JZTX-Ⅳ(IC₅₀=0.90±0.17μM) potently inhibits current on acutely isolated rat cardiac cell In addition,JZTX-Ⅳshows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons,differs from the classic site 3 toxins that do not affect the steady-state of inactivation.At high concentration,JZTX-Ⅳhas no significant effect on tetrodotoxin-resistant（TTX-R） sodium channels on rat DRG neurons and tetrodotoxin-sensitive（TTX-S） sodium channels on hippocampal neurons.2.HWTX-Ⅰis the most abundant toxic component in the venom of O.huwena.Sequence alignment analysis indicated that HWTX-Ⅰshares identical key residues with HNTX-Ⅳand HWTX-Ⅳ.In this paper,we investigated the action of huwentoxin-Ⅰ（HWTX-Ⅰ） purified from the venom of the Chinese bird spider Omithoctonus huwena on Ca²⁺,Na⁺ channels of adult rat dorsal root ganglion（DRG） neurons.The results showed that huwentoxin-Ⅰcould reduce the peak currents of N-type Ca²⁺ channels(IC₅₀≈100 nM) and TTX-S Na⁺ channels(IC₅₀≈55 nM), whereas no effect was detected on TTX-R Na⁺ channels.The comparative studies indicated that the selectivity of HWTX-Ⅰon Ca²⁺ channels was higher that of MVIIA and approximately the same as that of GVIA.HWTX-Ⅰis the first discovered toxin with the cross channel activities from the spider O.huwena venom similar toμO-conotoxins MrVIA and MrVIB.To ensure the cross channel activities was not because of poor purity,we synthesized HWTX-Ⅰand demonstrated that the sodium channel inhibition of synthetic HWTX-Ⅰ（sHWTX-Ⅰ） was similar in activity to natural HWTX-Ⅰon rat dorsal root ganglion（DRG） neurons.In addition,we tested the potent sodium channel inhibition of sHWTX-Ⅰon cockroach dorsal unpaired median（DUM） neurons (IC₅₀=4.80±0.58nM) and rat hippocampal neurons(IC₅₀=66.1±5.2nM). The further results showed that sHWTX-Ⅰ,over the IC₅₀ value,had no effect to steady-state activation and steady-state inactivation kinetics of sodium channels on DUM and hippocampal neurons as well as on DRG neurons.As the most abundant toxic component in the venom of O. huwena,the preference of HWTX-Ⅰfor insect DUM neurons suggests that it is the most important component in crude venom for prey paralysis.3.Using Reverse-phrase high performance liquid chromatography to isolate the O.hainana crude venom,there were 21 peaks.We collected all the peaks and tested 14 peaks.Three of them were found to inhibit the TTX-R sodium channels on DRG neurons.Further study,a novel toxin,designated Hainantoxin-Ⅶ（HNTX-Ⅶ） was isolated from the venom of Chinese spider Ornithoctonus huwena（Selenocosmia hainana）.HNTX-Ⅶis a 3830.23 Da peptide with 33 amino acid residues containing six conservative cysteines.The full-length cDNA of JZTX-Ⅳencodes an 85-amino acid precursor.Under whole-cell patch clamps conditions,HNTX-Ⅶcould reduced the peak current of TTX-S, TTX-R VGSCs on DRG neurons,TTX-S VGSCs on hippocampal neurons in rats and and TTX-S VGSCs on DUM neurons in cockroaches. The IC₅₀ was 0.169±0.046μM,0.676±0.049μM,1.10±0.09μM, 15.1±2.7nM,respectively.HNTX-Ⅶdid not affect steady-state activation curves and steady-state inactivation curves of VGSCs on DRG neurons.HNTX-Ⅶmade the steady-state activation curves of VGSCs shift to depolarization direction（10 mV） and steady-state inactivation curves of VGSCs shift to hyperpolafization direction （13.4mV） on hippocampal neurons.HNTX-Ⅶselectively affected the kinetics of sodium channel subtype,might be an important tool to explore the structure and functions relationship of voltage gated channels.HNTX-Ⅶalso inhibit the potassium currents on cockroach DUM neurons with the IC₅₀=13.7±1.2nM.更多还原