【摘要】 目的:以m5AChR-G11融合蛋白为工具,筛选m5受体亚型特异性配体,并且探讨m5AChR-G11融合蛋白中两组分相互作用的机制。方法:采用杆状病毒Sf 9细胞表达系统,制备m5AChR-G11融合蛋白;通过[3H]QNB放射配体结合实验和[35S]GTPγS替代结合实验,检测m5AChR-G11融合蛋白的功能。结果:m5AChR-G11融合蛋白的表达水平为(60.39±1.95)pmol.mg-1蛋白。不同配体存在时使融合蛋白中G11与GDP的亲和力发生变化,ace-tylcholine,M cN-A-343,AF-102B,R-(+)-hyoscyam ine,trop icam ide,alcuron ium和atrop ine存在时以及无配体时,GDP的IC50值分别是134.90,46.77,38.02,10.00,13.80,18.62,5.89,23.40μmol/L。结论:杆状病毒Sf 9细胞表达系统表达的m5AChR-G11融合蛋白具备m受体配体结合特性和两组分间的偶联功能。乙酰胆碱是m5受体的完全激动剂,M cN-A-343和AF-102B是部分激动剂,R-(+)-hyoscyam ine,trop icam ide,alcuron ium和阿托品是拮抗剂。更多还原

【Abstract】 Objective: To prepare m₅AChR-G₁₁ fusion protein as a tool to screen muscarinic ligands specific for m₅AChR,and to explore the mechanism of the effects between 2 partners in m₅AChR-G₁₁ fusion protein.Methods:m₅AChR-G₁₁ fused DNA was generated and then expressed in Baculovirus-Sf 9 cells.\QNB and \GTPγS binding experiments were performed to study the function of m₅AChR-G₁₁ fusion protein and to screen the ligands specific for m₅AChRs.Results:The expression level of m₅AChR-G₁₁ was（60.39±1.95） pmol·mg^-1 protein.The affinity of GDP to G₁₁ partner changed in the presence of different muscarinic ligands.The IC₅₀ values of GDP in the presence of acetylcholine（ACh）,McN-A-343,AF-102B,R-（+）-hyoscyamine,tropicamide,alcuronium,and atropine were 134.90,46.77,（38.02）,10.00,13.80,18.62,and 5.89 μmol·L^-1,respectively.The IC₅₀ value in the absence of muscarinic ligand was 23.44 μmol·L^-1.Conclusion:The m₅AChR-G₁₁ fusion protein has the pharmacological specificity of m₅ receptor and the efficient signaling of 2 partners.ACh is a full agonist of m₅AChRs.Mcn-A-343 and AF-102B are partial agonists of m₅AChRs.R-（+）-hyoscyamine,tropicamide,alcuronium,and atropine are antagonists of m₅AChRs.更多还原