Effects of various muscarinic ligands on M₂AChR-G_i1a fusion protein expressed in Sf9 insect cells  

不同的毒蕈碱配体对在Sf9昆虫细胞内表达的M2AChR-Gi1α融合蛋白的作用（英文）

作　　者：王昊[1] 郭政东[1] 李智[1] 刘洪瑞[1] 

机构地区：[1]中国医科大学药理学教研室

出　　处：《Acta Pharmacologica Sinica》2002年第3期42-48,共页中国药理学报（英文版）

基　　金：Project supported by the National Natural Science Foundation of China (№ 30171077) and Natural Sciencc Foundation of Liaoning Province (№ 002039).

摘　　要：To generate M2AChR-Gila fusion protein in baculovirus-Sf9 cells system and detect the effects of various muscarinic ligands and magnesium ion on the interaction of fused M2AChR and Gila. METHODS:M2AChR-Gila fused DNA was generated in a two-step polymerase chain reaction (PCR) and then expressed in Sf9 cells to produce fusion protein. [3H] L-quinu-clidinyl benzilate ( QNB ) and [35S] GTPγS binding experiments were performed to study the function of M2AChR-Gila fusion protein. RESULTS: The expression level of M2AChR-Gila was ( 20. 12±0.14 ) nmol·g-1 protein. The affinity of GDP to Gila partner changed in the’ presence of different muscarinic ligands. IC50 values (95 % confidence limit) of GDP in the presence of acetylcholine (ACh), carbamylcholine, (4-hydroxy-2-butyny1)-1- trimethylammonium- m - chloro-carbanilate chloride (McN-A-343), pilocarpine, and atropine were 178 (148 - 214)μmol/L, 158 ( 126 - 199) ,μmol/L, 66 (56-78) μmol/L, 62 (55 - 72) ,μmol/L, and 5.0 (4.6-5.5)μmol/L, respectively, and that in the absence of muscarinic ligand was 15.9 (14.3-17.6)μmol/L. Apparent affinity for GDP in the presence of carbamylcholine was markedly decreased with increasing MgCl2 concentrations, although the apparent affinity in the presence of atropine was not affected. CONCLU-SION; The M2AChR-Gila fusion protein has the pharmacological specificity of M2 receptor and the efficient signaling of the two partners. Affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands. Mgr2+ is necessary for the action of M2AChR on Gila.目的:表达M₂AChR-G_ila融合蛋白,检测不同配体或镁离子对M₂AChR与G_ila间相互作用的影响.方法:两步PCR建立M₂AChR-G_ila融合DNA,并在Sf9细胞内表达.[³H]QNB和[³⁵S]GTPγS结合实验检测M₂AChR-G_ila融合蛋白功能.结果:M₂AChR-G_ila的表达水平为（20.12±1.14）nmol·g^-1protein.不同配体的存在使融合蛋白中G_ila与GDP的亲和力 发生变化.乙酰胆碱,卡巴胆碱,McN-A-343,毛果芸香碱和阿托品存在以及无配体存在时,GDP的IC₅₀值（95％可信区间）分别是178（148-214）μmol/L,158 （126—199）μmol/L,66（56—78）μmol/L,62（55-72）μmol/L,5.0（4.6-5.5）μmol/L和15.9（14.3-17.6）μmol/L.在卡巴胆碱的作用下,C_ila随Mg²⁺浓度增加而减小对GDP的亲和力,而阿托品使G_ila不受Mg²⁺浓度的影响,始终保持对GDP的高亲和性.结论:杆状病毒-Sf9细胞系统表达的M₂AChR-G_ila具备M受体配体结合特性和组分间的偶联功能.M₂AChR-G_ila对GDP的亲和性决定于M₂受体配体的性质,分析GDP亲和力的大小有利于筛选和鉴别M₂亚型特异性药物.Mg²⁺离子是M₂AChR-G_ila与低亲和性GDP结合必需的阳离子.

关 键 词：muscannic receptors inhibitory G G-PROTEIN recombinant fusion proteins muscannic agonists muscannic antagonists adenosine triphosphate guanosine triphosphate guanosine diphosphate radio-ligand assay 

分 类 号：R965.1[医药卫生—药理学]