大电导钙激活(BKCa)钾通道被认为是具有电压和Ca2+双重门控特性的一类独特离子通道。BKCa的功能失调可导致一系列如癫痫等与神经元超兴奋性有关的神经疾病。迄今，BKCa通道亚型多样性、多亚基组装、结构异化等复杂机制的科学疑题仍有待破解。申请人拟借助来源我国特有产毒物种特异性BKCa通道毒素Martentoxin，采用分子生物学、计算生物学、药理学和生物大分子互作分析等手段，解析BKCa通道与Martentoxin相互作用的微结构域定位和关键氨基酸残基；同时，利用人工合成多肽或重组表达方法，以含有BKCa通道孔区的KcsA-BK为“诱饵”，借鉴配体与靶通道相互识别、药理结合、位点定位等关键信息，探讨设计优化亚型特异性BKCa通道毒素多肽的策略。以期验明BKCa通道上药理敏感性的新靶点，为研究BKCa通道多样性和抗癫活性药物设计提供“溯源性母本”性新思路。

Large-conductance Ca2+-activated K+ channel (BKCa channel) is a kind of ion channel uniquely gated by both [Ca2+]i and voltages. Dysregulation of BKCa channels are one of the important factors that contribute to epileptogenesis. The current study aims to investigate the molecular mechanisms of Martentoxin, a BKCa2+ channel-specific ligand,interaction with receptor sites by molecular biology, BiaCore T200 biomolecular interaction analysis and molecular modelling.Through constructing an in vitro expressed chimeric channel,KcsA-BK,which incorperated with K+ permeable pore of BK channel, the extracellular vestibule of the KcsA-BK may ‘trap’ the Martentoxins and thus the pharmacological kinetics,affinitive potency and precise localization of binding sites could be identified. Meanwhile,with molecular modelling, the possible recognition sites of BK channel-specific ligands will be predicted and verified for the optimized binding,which could help to design anti-epileptic peptides. This study could hopefully illuminate the novel pharmacological targets and provide ‘tracing origin’ based on natural toxins to probe the diversities of BK channels,which is likely to highlight path to develop active peptides for anti-epilepsy therapy.

本项目采用膜片钳、计算生物学、SPR和整体动物脑电等研究方法，以天然多肽毒素martentoxin和BK通道为研究对象，在模式动物、细胞和分子三个层面，揭示martentoxin可选择性地抑制神经型BK通道（α+β4），单（α）型和平滑肌型（α+β1）对martentoxin不敏感，BK通道跨膜区的Y294为识别martentoxin的关键性位点，D261和E276对于识别martentoxin并不重要；此外，β4亚基胞外环对神经型BK通道与martentoxin的互作也至关重要。通过改造pGEX-4T-3载体，重组表达了martentoxin，其活性与天然毒素一致。经鉴定K20， S24 和 Q26 是识别神经型BK通道的关键位点。Martentoxin可抑制由PTZ诱导的大鼠惊厥发作和复发，延长癫痫异常脑电的潜伏期，减少癫痫发作的时程，下调海马区c-fos的表达。以上结果提示了BK通道可作为癫痫治疗的新靶标，martentoxin及其模拟物具备作为抗癫痫药物分子的潜质。