应用m_5AChR-G_(11α)融合蛋白鉴别M_5受体亚型的特异性药物  

Identification of specific drugs for M_5 using m_5AChR-G_ (11α) fusion protein

作　　者：刘洪瑞[1] 郭政东[2] 韩雪松[2] 王海波[3] 李智[1] 

机构地区：[1]中国医科大学基础医学院天然药物研究室,辽宁沈阳110001 [2]中国医科大学基础医学院药理学教研室,辽宁沈阳110001 [3]中国医科大学基础医学院病理生理学教研室,辽宁沈阳110001

出　　处：《中国药理学通报》2006年第8期917-921,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30171077)

摘　　要：目的采用Sf9细胞系统表达m5AChRG11α融合蛋白,通过检测GDP与m5AChRG11α融合蛋白的亲和力大小来鉴别m5AChR的特异性激动剂和拮抗剂。方法用两步PCR反应重组m5AChRG11α融合蛋白cDNAs,并插入到pBacPAK9病毒载体中,利用Sf9细胞表达m5AChR受体蛋白和m5AChRG11α融合蛋白,通过[3H]QNB结合饱和实验及[35S]GTPγS竞争性替代结合实验,检测受体表达水平及GDP与m5AChRG11α融合蛋白的亲和力。结果m5AChRG11α融合蛋白表达水平是(47.6±3.2)nmol·g-1膜蛋白。不同配体的存在使融合蛋白中G11α与GDP的亲和力发生变化。ACh、YM796、Oxotremorine、Methixene、Dextimide及atropine存在以及无配体存在时,GDP的IC50值分别是128.0,72.1,68.5,16.2,14.9,9.7和20.8μmol·L-1。结论Sf9细胞系统表达的m5AChRG11α融合蛋白具备M受体配体结合的特性和组分间偶联功能。m5AChRG11α融合蛋白对GDP的亲和性决定于M受体配体的性质。对于m5AChRG11α融合蛋白,ACh是m5AChR完全激动剂,YM796和Oxotremorine是部分激动剂;atropine,Methixene和Dextimide是拮抗剂。Aim The m5AChR-G11α fusion protein was expressed by baculovirus-St9 cells system, then using it to identify the specific agonists and antagonists for m5AChR via detecting the affinity of GDP and m5AChR-G11α. Methods The m5AChR-G11α, fused cDNAs were generated via a two-step PCR protocol and inserted into pBacPAK9 virus vector. We expressed m5AChR-G11α, fusion protein and m5AChR protein using baeulovirus-St9 cell system. [ ^3H ] QNB and [ ^35 S ] GTPγS binding tests were performed to detect the expressional level of receptor proteins and determine the affinity of GDP and m5AChR-G11α, fusion protein. Results The expression level of m5AChR-G11α, was （47.6 ±3.2） nmol · g^-1 protein. The affinity of GDP to GDP, partner changed in the presence of different muscafinic ligands. IC50 values of GDP in the presence of ACh, YM796, Oxotremorine, Methixene, Dextim-ide and atropine were 128.0, 72. 1, 68.5, 16. 2, 14. 9 and 9.7 μmol· L^-1 respectively, and that in the absence of muscarinic ligand was 20. 8 μmol · L^-1. Conclusion The m5AChR-G11α, fusion protein has the pharmacological specificity of M5 receptor and the efficient coupling interaction of the two partner. Affinity of GDP to ligand bound m5AChR-G11α, fusion protein represents the species of muscarinic ligands. ACh is a full agonist for m5AChR-G11α, fusion protein, YM796 and oxotremorine are partial agonists, while methixene, dextimide and atropine are antagonists.

关 键 词：毒蕈碱乙酰胆碱受体 G蛋白 融合蛋白 激动剂 拮抗剂 

分 类 号：R341[医药卫生—基础医学] R341.3