基于新激活剂壳寡糖的TMEM16A离子通道门控机制研究

CaCCs (Calcium-activated Chloride Channels) or its molecular counterpart TMEM16A (Transmembrane 16A) participate in many vital biological processes, including cell volume regulation, neuronal and cardiac excitability, smooth muscle contraction and transendothelial ion/fluid transport. TMEM16A ion channel is potential drug target for hypertension, asthma, gastrointestinal motility disorders, cystic fibrosis, etc. So screening novel channel regulators has become a hot issue. But how the CaCCs/TMEM16A ion channel opening “door”(also called gating mechanism) remains a major obstacle. On the basis of the novel TMEM16A activator chitosan oligosacchraride (COS) discovered by the applicant, this proposal will focus on the TMEM16A activating mechanisms by COS through a biological experiment and molecular dynamics calculation combined strategy. .First, three different steps are carried out to confirm the detailed activation mechanism, in which, (a) by comparison with other chloride ion channels such as CFTR, CLC, the specificity of COS to TMEM16A is confirmed; (b) Synchronous recording differently fluorescent-labeled COS and intracellular free calcium ions is performed to study real time activation progress of COS to TMEM16A channel with Laser Scanning Confocal Microscopy; (c) the effect of COS on the smooth muscle contraction of small intestine is assayed to explore its physiological implications. .Second, the molecular structure of the best monomer of COS is identified by comparing different degrees of polymerization and substituent groups among COS monomers and the corresponding analogues. So the required chemical group and molecular size would be obtained for the subsequent research..Third, the coarse binding domain of COS and TMEM16A ion channel molecule is located by static electricity potential calculation. Then, the three dimensional structure of the binding domain is constructed by homology modeling method, which is to molecularly docked with the best COS molecule to characterize the micro-interaction between COS and TMEM16A. Next, the key amino acid in TMEM16A channel is to be site-directed mutated and the resulted mutant will be checked with patch clamp experiments. .This proposal would clarify the dynamical gating process of CaCCs/TMEM16A, identify the pivocal chemical structure of CaCCs' activating molecules, which would lay the theoretical foundation for CaCCs-targeted medicine design.

钙激活氯离子通道CaCCs/TMEM16A可调节多种基本生理过程，其功能受损将导致多种疾病，其调节剂筛选和药理研究已成为热点问题。然而该通道门控机制不明严重制约以上研究。在申请人前期发现壳寡糖（COS）为TMEM16A通道激活剂的基础上，项目拟将生物学实验与分子动力学计算方法相结合，研究COS激活TMEM16A通道的作用机制，内容包括：1.通过与其他氯离子通道相比较，研究COS激活作用特异性；显微成像实验研究激活作用直接性；研究COS对小肠平滑肌收缩反应的影响。2.通过与结构类似物比较，研究聚合度和取代基团的影响，找出激活作用最优COS单体分子结构。3.静电势分析粗筛COS与TMEM16A通道互作结构域，将其同源模建三维结构与最优COS单体分子对接，研究二者相互作用细节，定位通道分子上关键氨基酸位点，并用实验检验。项目将揭示TMEM16A通道的门控机制，为相关疾病靶向药物分子设计提供依据。

TMEM16A/CaCCs 表达异常与多种疾病相关,其调节剂筛选及药理学研究已成热点问题。本项目在前期发现壳寡糖为TMEM16A离子通道激活剂的基础上展开激活机制研究。经过项目组全体成员的积极努力，在International Journal of Biological Macromolecules, Pflügers Archiv-European Journal of Physiology，Journal of Membrane biology等国际知名学术期刊上，发表论文7篇，总影响因子：15.512，其中JCR分区一区论文1篇，二区论文2篇。申请国家发明专利4项，目前已获得授权发明专利2项。项目组成员陈娅斐和李军委分别获得河北省优秀博士学位论文荣誉称号，项目负责人陈娅斐获得河北省青年拔尖人才荣誉称号及项目资助。.项目原定研究计划圆满完成，并进行了延伸性研究。具体工作包括：.实验方面：用膜片钳内面向外和全细胞模式的电生理学实验确认壳寡糖对于TMEM16A通道的激活作用及其作用特异性，并与钙离子对其激活效力作比较。利用激光共聚焦显微镜进行YFP荧光淬灭实验实时观察壳寡糖作用下TMEM16A通道的开放过程，研究了壳寡糖进入细胞并激活通道的微观作用机制。.理论方面：应用同源模建方法构建TMEM16A离子通道的三维结构，利用全原子分子动力学方法对壳寡糖单体分子与TMEM16A通道之间的弱相互作用进行模拟，预测关键化学基团和关键氨基酸，并结合实验手段，比较了通道与钙离子的结合方式和通道与壳寡糖分子结合方式之间的关系，系统分析了壳寡糖激活TMEM16A通道的作用机制，圆满完成了项目的研究任务。.在此基础上，我们利用改进了的筛选方法，又鉴定了多种新的TMEM16A的天然产物分子激活剂与抑制剂——β-1,3-葡聚糖、白藜芦醇和人参皂苷Rb1，利用理论与实验结合的策略分析了关键作用基团，并在改善胃肠动力和抗肿瘤两方面进行生理水平的探究，为下一步将目标化合物改造成具有高效、低毒、高靶向性药物打下坚实基础。