不同配体对m_5乙酰胆碱受体-G蛋白亚单位G_(11)融合蛋白表达和功能的影响  

Effects of various muscarinic ligands on the expression and function of m_5 muscarinic acetylcholine receptor-G_(11) fusion protein

作　　者：郭政东[1] 王海波[2] 李智[1] 刘洪瑞[1] 郭政礼[3] 赵立斌[3] 

机构地区：[1]中国医科大学药理学教研室,辽宁沈阳110001 [2]中国医科大学病理生理学教研室,辽宁沈阳110001 [3]沈阳市中心医院药剂科,辽宁沈阳110024

出　　处：《中国药理学与毒理学杂志》2004年第1期11-16,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目 (3 0 1710 77);辽宁省自然科学基金资助项目 (0 0 2 0 3 9)~~

摘　　要：目的　制备毒蕈碱乙酰胆碱受体m5亚型与G 蛋白亚单位G1 1 (m5AChR G1 1 )融合蛋白 ,探索m5AChR与G1 1 之间的耦联功能、相互作用及其影响因素。方法　两步PCR法建立m5AChR与G1 1 融合cDNAs,并在Sf9细胞内表达。 [3 H]QNB和 [3 5S]GTPγS结合实验检测m5AChR与G1 1 融合蛋白的功能。结果m5AChR G1 1 融合蛋白的表达水平为 (60 .4± 2 .0 )nmol·g-1 膜蛋白。不同配体存在使融合蛋白中G1 1 与GDP的亲和力发生变化。乙酰胆碱(ACh)、卡巴胆碱、毛果芸香碱、异丙铵、异丙嗪及阿托品存在时 ,GDP的IC50值分别为 1 35,63 ,1 82 ,4.1 ,8.9和 5.9μmol·L-1 ,无配体存在时 ,GDP的IC50 值为 2 3.4μmol·L-1 。结论　杆状病毒Sf9细胞系统表达的m5AChR G1 1 融合蛋白具备m受体配体结合特性和组分间耦联的功能。m5AChR G1 1 融合蛋白对GDP亲和力取决于m受体配体的性质 。AIM To test the couping function, the interreaction and the influence factors between m 5 muscarinic acetylcholine receptor(m 5AChR) and G 11 protein by generating m 5AChR and G 11 fusion protein(m 5AChR G 11 ) in baculovirus Sf9 cells system. METHODS m 5AChR G 11 Fused DNA was generated in a two step PCR and then expressed in Sf9 cells to produce fusion protein. QNB and [γ 35 S]GTP binding experiments were used to study the function of m 5AChR G 11 fusion protein. RESULTS The expression level of m 5AChR G 11 was (60.4± 2.0) nmol·g -1 protein. The affinity of GDP to G 11 partner changed in the presence of different muscarinic ligands. IC 50 values of GDP in the presence of acetylcholine(ACh), carbachol, pilocarpine, isopropamide, promethazine and atropine were 135, 63, 182, 4.1, 8.9 and 5.9 μmol·L -1 , respectively, and that in the absence of muscarinic ligand was 23.4 μmol·L -1 . CONCLUSION The m 5AChR G 11 fusion protein expressed in baculovirus sf9 cells system has pharmacological specificity for m 5 receptor and the efficient coupling function between the two partners. Affinity of GDP to ligand bound fusion protein represents the species of muscarinic ligands. This is helpful to screen and detect new specific ligands of the muscarinic receptors subtype.

关 键 词：配体 毒蕈碱 G蛋白类 融合蛋白 胆碱能激动剂 胆碱能拮抗剂 乙酰胆碱受体 

分 类 号：R962[医药卫生—药理学]