一种在胞内调控钠通道活性的多肽毒素的作用机制研究

Peptide toxins play important roles in the defense and prey capture of venomous animals. They are also useful and potential for relative basic researches and new drug development, which has attracted great attention from life sciences, medicine and pharmaceutical fields. Great progress has been made in peptide toxin researches, but most of them remain unclear. Therefore, screening and identification of new peptide toxins, new mechanism investigation and application of peptide toxins will be the most important issue in this field. In our previous study, we identified a novel peptide toxin from centipede venom. It contains 55 amino acid residues and 4 pairs of disulfide bonds, showing new amino acid sequence and space structure. Moreover, it is able to enter into cell plasma and delay the fast inactivation of sodium channels in the intracellular side. This is a new mechanism of peptide toxins regulating sodium channel activity. In this study, in order to elucidating the mechanism of the peptide toxin delaying the fast inactivation of sodium channels, we will continue to study electronical property of this toxin regulating sodium channels, structure and function relationship, the pathway of the toxin penetrating into cells and the molecular mechanism of the toxin and sodium channel interaction. The results of this study will provide insight of biodiversity and action mechanisms of peptide toxins from venomous animals, and also offer new understanding and new useful tool for the study of sodium channel inactivation in the intracellular side.

多肽毒素对有毒动物的防御和猎食具有重要作用，并在相关基础研究和新药研发等方面展现了很大的应用价值，获得生物、医学和制药行业的密切关注，研究进展迅速，但相对于多肽毒素的多样性和复杂性，目前所得仍是冰山一角，新毒素的筛选和鉴定、新作用机制的发现和阐明、高效利用等仍是多肽毒素研究的重要方向。本项目前期研究从蜈蚣毒液鉴定了一个作用于电压门控钠通道的多肽毒素rpTx1，含55个氨基酸残基和四对二硫键，具有全新的氨基酸序列；更重要的是，它可进入细胞内并在胞内抑制钠通道失活，是一种多肽毒素调控钠通道活性的新机制，故值得深入探究。本项目拟在电生理机制、结构与功能关系、入胞机制及胞内与钠通道相互作用分子机制四个层次开展研究，全面揭示rpTx1在胞内调控钠通道活性的新作用机制。本项目的完成将扩展和深化人们对多肽毒素分子多样性和作用机制等的理解，为钠通道失活的胞内调控提出新见解及研究切入点和分子工具。

电压门控钠通道是动作电位产生和传导的基础，在神经系统、心脏起搏和肌肉收缩等重要生理过程中扮演至关重要的作用。来源于天然动物毒液的神经毒素是研究钠通道结构与功能不可或缺的分子工具。本项目从蜈蚣毒液中鉴定到一个可进入细胞内抑制钠通道快失活的多肽毒素rpTx1，这与先前发现的钠通道多肽毒素具有完全不同的作用模式，采用的是一种全新的作用机制。通过膜片钳电生理、基因突变、SPR、细胞免疫荧光、双循环突变和小鼠动物模型等研究手段系统得到如下研究结果：1.揭示了rpTx1具有穿膜和抑制钠通道快失活两个相对独立的功能结构域；2.阐明了rpTx1与钠通道相互作用的分子机制，rpTx1与钠通道失活球IFMT区域相互作用，揭示钠通道固有的快失活门是一个新的神经毒素结合位点；3.利用rpTx1为探针，阐明钠通道失活球IFMT位置与VSD II的状态转换相关联，揭示它们变构调节通道的激活和快失活;4.阐明了rpTx1的生物学效应。本项目研究扩展和深化了人们对有毒动物多肽毒素作用机制等的理解，为钠通道失活的胞内调控提出新见解及研究切入点和分子工具。