KCNT1通道在氟烷类麻醉剂和丙泊酚全麻效应中不同作用的机制研究

Research on receptors or ion channels targets of general anesthetics is a hot field of general anesthesia studying. Our preliminary work found the KCNT1 potassium ion channel plays different roles in the process of general anesthesia induced by halothane class A narcotics and propofol. Based on this preliminary data, we will identify the role of KCNT1 channels played during the process of general anesthesia induced by different general anesthetics by comparing the effects of general anesthetics on wild type mice, KCNT1 KO mice and KCNT1 over-activity mice. We will investigate the change of neural excitability, synapse transmission induced by different general anesthetics by using behaviour, morphological and electro-physiological methods. This project also plans to address the neuronal nucleus in which KCNT1 channels play their roles by taking advantage of stereotactic injection KCNT1 KO mice with lenti-virus that carried KCNT1 channel gene. In the meantime, we will also address the Cl- binding site of KCNT1 channel by using molecular mutation and electrophysiology method. Also more experiments will be done to dissect the functional interaction of GABAa receptor and NMDA receptor with KCNT1 channels. Taken together, this project combined methods in vivo and in vitro to dissect the mechanism of KCNT1 channel that are involved in general anesthesia process induced by different general anesthetics. This project will increase our understanding of principles of general anesthesia and is helpful for developing new general anesthetics.

全麻药物受体和离子通道靶标研究是麻醉学研究领域的热点。我们前期工作利用KCNT1基因敲除的小鼠发现KCNT1钾通道在氟烷类麻醉药物和静脉麻醉药物丙泊酚的全身麻醉效应中起不同的作用。本课题以此为依据，通过行为学、形态学和电生理研究比较分析不同全麻药物在野生型小鼠、KCNT1通道基因敲除小鼠和KCNT1通道突变过度激活小鼠中的效果，揭示KCNT1通道参与不同全麻药物作用过程中神经元兴奋性、突触传递变化的异同。我们也将运用表达KCNT1通道的慢病毒核团定位注射方法来辨明KCNT1通道参与全麻的中枢核团。同时，通过分子突变和电生理实验解构 KCNT1通道的氯离子结合位点，并阐明KCNT1通道与GABAa受体、NMDA受体功能上相互作用的分子机制。综上所述，本课题结合在体和离体的方法阐释KCNT1通道在不同全麻药物作用过程中的机制，并以此深化对全身麻醉原理的理解，为发展新的全麻药物提供帮助。

本研究基于本实验室前期数据发现KCNT1基因敲除的小鼠对丙泊酚麻醉产生拮抗但对七氟烷的诱导敏感的现象而申请立题研究并获得批准。通过四年的研究，本课题首先通过形态学的研究明确了KCNT1基因表达丰富的核团，通过使用病毒载体将KCNT1基因表达回KCNT1基因敲除的小鼠的梨状皮质、杏仁核、中脑室旁核和网状核等核团，发现KCNT1基因敲除小鼠对丙泊酚的拮抗作用主要是通过提高室旁核兴奋性神经元的兴奋性机制实现。而对七氟烷的敏感性则是由于提高了网状核抑制性神经元的兴奋性而实现的。RNAscope 的数据显示，KCNT1通道在兴奋性神经元和抑制性神经元都有表达。电生理实验表明Slack通道敲除可降低其表达的神经元的动作电位阈值，增加产生动作电位的次数。而其gain-of-function的突变则产生相反的效果。这些结果阐明了KCNT1通道基因敲除小鼠对不同麻醉药物的敏感性不同的机制。同时，通过一系列的突变的筛查，我们鉴定出KCNT1通道的两个新的钠离子的结合位点和一个氯离子结合位点。这为进一步理解通道的生物物理性质和功能的关系奠定基础。通过一系列的研究，我们获得授权专利三项，发表Sci论文四篇，还有一篇在审稿中，另一篇论文在撰写投稿中。