烟碱型乙酰胆碱受体β4亚基点突变体的构建  

Construction of Nicotinic Acethlcholine Receptor β4 Subunit Mutant

作　　者：张璐 朱晓鹏[1] 长孙东亭[1] 罗素兰[1] ZHANG Lu;ZHU Xiaopeng;ZHANGSUN Dongting;LUO Sulan(College of Marine,Hainan University/Ministry of Education Key Laboratory for Tropical Biological Resources/Hiakou Key Laboratory for Marine Drugs,Haikou,Hainan 570228,China)

机构地区：[1]海南大学海洋学院/热带生物资源教育部重点实验室/海口市海洋药物重点实验室

出　　处：《热带生物学报》2019年第4期307-313,共7页Journal of Tropical Biology

基　　金：国家自然科学基金项目(81872794);海南省研究生创新科研课题(Hys2017-58)

摘　　要：利用PCR介导的定点突变技术构建包含β4亚基的烟碱型乙酰胆碱受体点突变体模型,同时采用双电极电压钳技术,对激动剂ACh和拮抗剂α-CTx RegIIA对突变受体的活性进行检测。结果表明:β4亚基胞外N端序列第168位的异亮氨酸(Ile)成功突变成了丝氨酸(Ser),突变后的受体对激动剂ACh的活性降低,对拮抗剂α-CTx RegIIA的活性降低了1.4倍,半阻断剂量(IC50)为170 nmol·L^-1。To explain the molecular mechanisms of interaction between β4 nAChR subunit and ligand(agonist ACh and inhibitor α-CTx RegIIA), a mutant of β4 nAChR subunit was constructed by substituting 168 th Ile(I) with Ser(S) in the Loop F of N-terminal ligand-binding region, by using the method of site-directed mutagenesis that PCR mediated. The results showed that the inward current of oocytes injected with cRNA of α3β4[S168 I] nAChR could be recorded by two-electrode voltage clamp. The mutant receptor had 1.4 times lower sensitivity to α-CTx RegIIA with a higher IC50 of 170 nmol·L^-1, compared with the wild-type α3β4 nAChR.

关 键 词：烟碱型乙酰胆碱受体 β4亚基 PCR介导的定点突变 双电极电压钳技术 

分 类 号：Q7[生物学—分子生物学]