蝎毒多肽martentoxin靶向大电导钙激活钾通道（BK）的抗癫痫机制研究

Epilepsy is a brain dysfunction syndrome caused by abnormal discharge of neurons. So far available drugs and surgical treatments for epilepsy suppress the symptoms temporarily with harmful side-effects. The abnormal gene expression and hyperfunction of BK channels are currently considered as the key factors causing the neuronal hyperexcitability as well as seizures. Therefore, it could be regarded as a strategy for alleviating seizures to discover the inhibitors of BK channels from natural products. Scorpion is the classic drug of traditional Chinese medicine for epilepsy treatments, of which scorpion venom is the active ingredient. It was found that the scorpion venom and extracted polypeptide martentoxin could effectively inhibit the activity of BK channels. Based on the results above, it is speculated that martentoxin inhibited neuronal hyperexcitability as well as seizures by targeting neuronal BK channels in vivo. The project aims to investigate the following subjects by electroencephalogram (EEG), patch clamp recording and computer simulation: (1) Dose-dependent effects of martentoxin on relieving kindling epilepsy model as well as temporal lobe epilepsy model; (2) Inhibition of martentoxin on the action potentials and binding kinetics of martentoxin with BK channel; (3) Key sites of martentoxin for recognizing BK channels. The project will not only in-depth analyze the anticonvulsant mechanism of martentoxin, but also provide novel molecular template and theoretical basis for the design of anti-epileptic drugs.

癫痫是一种神经元异常放电引起的脑功能失调综合征，现有药物的疗效有限。研究表明BK通道基因表达异常和功能亢进是引起神经元超兴奋和癫痫发作的关键因素，因此开发BK通道的特异性抑制剂不失为缓解癫痫发作的策略。全蝎是中医用于治疗癫痫的经典药物，蝎毒是其活性成分。申请人前期实验发现蝎毒及其多肽单体martentoxin可有效抑制BK通道的活性。基于上述提示，推测martentoxin在体内可能通过靶向抑制神经元BK通道，降低海马神经元兴奋性，缓解癫痫症状。本申项拟采用脑电记录、膜片钳和计算机模拟等手段检测：（1）martentoxin缓解点燃癫痫和颞叶癫痫模型惊厥发作的剂量效应；（2）martentoxin对神经元动作电位发放的抑制效应、与BK通道的结合动力学；（3）martentoxin识别BK通道的关键位点，深入解析martentoxin抗惊厥的机制，为抗癫痫药物设计提供新的分子模板与理论依据。

祖国医学治疗顽固性癫痫具有明显优势，已使用千年。全蝎是中医治疗癫痫的经典药物，项目负责人在青年基金的研究中，发现了martentoxin等多个全蝎抗癫痫活性提取物，并运用戊四唑癫痫大鼠模型、BK通道敲除小鼠，结合动物行为学、电生理学及结构生物学等技术，揭示：1）martentoxin可有效延长大鼠癫痫发作的潜伏期，减少发作的总时程，抑制癫痫大发作的次数、降低癫痫的死亡率；2）通过多电极脑电记录，观察到martentoxin可显著抑制癫痫大鼠海马脑区各频率场电位的功率谱密度，尤其是低频场电位；3）免疫组化显示，martentoxin减少了海马区兴奋性标志物cfos在神经元的表达；经尼氏染色发现，martentoxin治疗组的大鼠海马区神经元死亡情况显著低于对照组。提示martentoxin在癫痫发生过程中，有神经元保护作用；4）膜片钳检测发现，martentoxin可有效减少海马锥体神经元动作电位发放的次数，增大动作电位的宽度、间隔长度，后超级化电位的幅值和激活时间；5）在分子水平，martentoxin可以显著地抑制癫痫相关BK通道（α+β4）的活性。采用NMR解析了martentoxin与β4亚基复合体的结构和相互作用信息。经药理学实验验证，β4亚基的E104，E128，E122位点是识别martentoxin的关键氨基酸残基，martentoxin的K20，S24，S12，Q26是结合β4亚基的关键位点；6）延续性实验发现了一种抗癫痫发生的组分BmP02，BmP02干预可有效缓解小鼠癫痫自发的持续时间和发作级数，并抑制小胶质细胞的迁移能力和炎症因子IL-1β的表达。本项目初步解析了全蝎“熄风止痉”治疗癫痫发作的物质基础，为抗癫痫药物设计贡献了可能的分子模板，也为后续抗癫痫发生的课题提供了新的研究方向。

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