摘要
从东亚钳蝎蝎毒中分离鉴定出新型的N
东亚钳蝎(Buthus martensii Karsch,BmK)作为传统医药用于脑卒中、癫痫、抽搐等疾病的治疗已经有几千年的历
电压门控N
截至目前,东亚钳蝎蝎毒中已报道有至少34条α毒素或者同源多
最近的研究表明,东亚钳蝎粗毒可以通过直接增强Nav1.7的活性产生致痛作
SephadexG-50(美国GE Healthcare Life Sciences公司);三氟乙酸(TFA,色谱级,上海罗恩试剂);乙腈(CAN,德国Merck Drugs&Biotechnology公司);KCl、MgCl2、CaCl2(分析纯,南京化学试剂股份有限公司);Lipofectamin
AKTA蛋白纯化系统、XK26-1000色谱柱(美国GE Healthcare Life Sciences公司);Milli-Q Biocel超纯水仪(美国Millipore公司);Nanodrop 1000紫外可见光分光光度计(美国Thermo公司);C18反向色谱柱(4.6 mm × 250 mm,5 μm,大连依利特分析仪器有限公司);E2695高效液相色谱仪(美国Waters公司);落地式高速离心机(德国Beckman Coulter公司);pH计(上海仪迈仪器科技有限公司);Olympus倒置显微镜(日本奥林巴斯公司);MF-830电极抛光仪(日本Narishige公司);火抛光电极(美国World Precision Instruments公司);水平电极拉制仪P97、右手自动微操MP-225(美国Sutter公司);数模转换器Digidata 1550、单探头超低噪声膜片钳放大器Axon 200B(美国Molecular Devices公司);ALA-VM8全自动灌流系统(美国ALA公司);Von frey电子机械痛检测仪(美国IITC公司)。
大鼠Nav1.8表达质粒(rNav1.8)由上海生命科学研究所鲍岚研究员赠送;东亚钳蝎(产地陕西,经南京师范大学戴建华教授鉴定为东亚钳蝎);人N
利用电刺激法提取东亚钳蝎毒液,具体方法参考文献[
将经HPLC纯化后冻干的P12用去离子水溶解后进行15% SDS-PAGE电泳,之后将目的蛋白条带切割,然后利用蛋白回收试剂盒回收多肽。多肽样品利用飞行时间质谱检测相对分子质量,然后采用岛津生物技术蛋白质组学试剂盒按照标准流程进行还原、烷基化、酶解,最后酶解肽段经MonoTip C18脱盐后浓缩上样,并利用岛津激光生物实验室MALDI校准套件进行校正。肽质量指纹图谱信号(PMF)用蛋白组学质谱分析软件Mascot上传至蛋白质序列数据库SWISS-PROT进行肽段序列的分析,完成蛋白鉴定。N末端氨基酸测序参考本课题组前期工作进
同源模建参考使用Accelrys Discovery Studio 4.1软件,并利用BmK M1的。晶体结构(PDB ID:1SN1)作为模板对BmK M2进行模型构建。然后对最佳同源模型进行MD模拟,在AMBER 12软件中优化得到模拟最佳的3D结构,详细步骤见文献[
BmK M2的电生理活性在hNav1.7-HEK293及rNav1.8-ND7/23细胞上检测。所使用的细胞浴液及电极内液配方参考本课题组前期工作配
G = Gmax/{1 + exp[(V1/2, act -V)/k]}
其中Gmax是最大Na电导,V1/2, act是50% N
对于稳态失活实验,细胞钳制在-120 mV,通过一系列持续500 ms的脉冲电压刺激(-100至 -10 mV,以10 mV为增量)实现稳态快速失活,其余未失活的通道通过40 ms步进去极化激活至-10 mV。快速失活曲线以玻尔兹曼方程拟合得到。I/Imax = Rin + (1-Rin)/{1 + exp[(V - V1/2, inact)/k]},其中Imax是最大Na电流V1/2, inact是50% N
对于关闭状态下的快速失活曲线,刺激方案参考Herzog
选用野生型C57BL/6J雄性小鼠(6周,20 ± 2 g),共3组,每组5只,足底(左后肢注射体积10 μL)分别注射BmK M2(50 nmol/kg)7 μg和BmK M2(50 nmol/kg) 7 μg + GpTx-1(5 nmol/kg)。选用Nav1.8 KO雄性小鼠(6周,20 ± 2 g)5只,足底注射BmK M2(50 nmol/kg)7 μg。统计足底注射后30 min内小鼠舔足和抖足的次数。小鼠舔足实验结束后,当疼痛行为(舔足、抖腿、悬腿)明显减少或消失,将待测小鼠置于金属网格上适应5 ~ 10 min,然后用Von frey电子机械痛检测仪对给药足底机械阈值进行测试,当小鼠受到机械针刺激后会产生自然缩足(迈步不计算在内)时的力量阈值为其缩足阈值(以质量单位克计算),每只小鼠共重复测试5次,并以平均值作为最终参考值。
为了分离东亚钳蝎蝎毒中的多肽,首先利用SephadexG-50分子筛对提取的粗毒进行了分离。然后进一步对SephadexG-50分子筛的P7峰进行了HPLC分离。HPLC图谱中显示有23个峰,其中峰形完整且可能为高纯度单体的有14个峰(

Figure 1 Purification and identification of BmK M2
A: RP-HPLC analysis of P7 from sephadex G-50 gel filtration column chromatography (P12 marked by red circular); B: BmK M2 mass spectrometry identification and peptide mass fingerprinting identification; C: The amino acid sequence of Peptide of BmK M2. The disulfide bond pair is marked with a box, and the red letters is the N-terminal sequencing result
为进一步明确BmK M2的结构并为其潜在生物学活性探索提供依据,将BmK M2与多种已知N

Figure 2 Homology modeling and molecular dynamics of BmK M2
A: Sequences alignment showed high similarity between BmK M2 and BmK M1,BmK M3,BmK M4,BmK M8,OD1,AaH2; B: Homology modeled structure of BmK M2
Nav1.7表达于外周神经,是与疼痛信号传递最为密切的N

Figure 3 Effect of BmK M2 on Nav1.7 expressed in HEK293 cells
A: Currents from HEK293 cells expressing Nav1.7 channels in the absence and presence of 1 μmol/L BmK M2 elicited by repeated pulses (Vh = -120 mV, scale bars: 2 nA, 5 ms); B: Current-voltage (I-V) curves before (black) and after (red) treatment of 1 μmol/L BmK M2; C: BmK M2 alert the inactivation properties of Nav1.7
进一步研究了BmK M2对Nav1.7的激活和稳态失活曲线的影响。电生理结果显示,BmK M2在-140 mV至-70 mV期间加速失活,在-70 mV至0 mV期间延迟失活(

Figure 4 Effect of BmK M2 on the activation and steady-state fast inactivation curves of Nav1.7 channels
A: Currents from HEK-Nav1.7 cells expressing Nav1.7 channels in the absence and presence of 1 μmol/L BmK M2 elicited by repeated pulses (Vh = -120 mV, scale bars: 2 nA, 5 ms); B: Voltage-dependence of steady-state fast inactivation curves before (black) and (red) after treatment of BmK M2 (Vh =-120 mV, scale bars: 2 nA, 5 ms); C: Voltage-dependence of activation curves before (black) and after (red) treatment of BmK M2. Steady-state inactivation was estimated by measuring the cell conductance elicited by 20 ms test pulses to 0 mV after 500 ms prepulses to potentials over the range of -140 mV to -10 mV
电压门控离子通道的失活是内在的自动调节过程,是控制动作电位的发生和形状以及在可激发组织中建立发射模式所必需

Figure 5 Effect of BmK M2 on the closed state fast inactivation of Nav1.7 channels
A and B: Representative raw traces of N
Nav1.8是表达在外周感觉神经元上的另一种主要的TTX-R N

Figure 6 Effect of BmK M2 on Nav1.8 expressed in ND7/23 cells
A: Currents from ND7/23 expressing Nav1.8 channels in the absence and presence of 1 μmol/L BmK M2 elicited by repeated pulses (Vh = -120 mV, scale bars: 2 nA, 5 ms); B: Current-voltage (I-V) curves before (black) and after (red) treatment of BmK M2; C: Voltage-dependence of activation curves before (black) and after (red) treatment of BmK M2
BmK M2抑制Nav1.7的失活将会降低动作电位发放阈值,从而诱发疼痛行为。因此,进一步测试了BmK M2的致痛活性。结果提示,在野生型C57BL/6J(WT)小鼠后爪注射BmK M2后可引起剧烈的疼痛行为和机械痛敏感(

Figure 7 BmK M2 evoked pain response is abolished by Nav1.7 specific blocker ()
A: Intraplantar injection of BmK M2 (50 nmol/kg) or BmK M2 (50 nmol/kg) + GpTx-1 (5 nmol/kg) in C57BL/6J mice, and BmK M2 (50 nmol/kg) in Nav1.8 KO mice, measured as the number of paw flinches per 5 min after injection. B: Total number of paw flinches within 30 min following the intraplantar injection of BmK M2 in mice. C: Effects of BmK M2 on mechanical hypersensitivity were evaluated using the paw withdrawal threshold.BmK M2 significantly reduced the paw withdrawal threshold, which was inhibited by GpTx-1
本研究采用Sephadex G-50分子筛和高效液相色谱技术从陕西东亚钳蝎蝎毒中纯化出蝎毒组分P12。利用多肽指纹图谱和氨基酸测序技术发现蝎毒组分P12的氨基酸序列与基于晶体学分析预测的BmK M2序列一
Nav1.7和Nav1.8是外周神经中传递疼痛信号的两个主要N
本研究结果表明,BmK M2可作为一种新型的N
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