摘要
目的表达m2-Gilα融合蛋白,通过检测GDP与m2-Gilα融合蛋白的亲和力大小来鉴别m2受体的特异性激动剂、拮抗剂和反向激动剂。方法用两步PCR反应重组m2-Gilα融合蛋白cDNAs,并插入到pBacPAK9病毒载体中,利用Sf9细胞表达M2受体蛋白和m2-Gilα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m2-Gilα融合蛋白的亲和力。结果m2-Gilα融合蛋白表达水平是(18.14±0.17)pmol·mg(膜蛋白)-1。m2-Gilα融合蛋白与[3H]QNB的结合量随着[3H]QNB浓度的升高而增加,10 μmol·L-1GTDγS使m2-Gilα融合蛋白与[3H]QNB的饱和结合曲线明显下移。Ach使m2-Gilα融合蛋白与[35S]GTPγS竞争性替代结合曲线明显右移,Pilo稍次之,alcuronium,AF—DX116,Iso及atropine 则使曲线左移,GDP的IC50值分别是168.4,152.3,6.84,6.12,5.59,4.52 μmol·L-1,无配体存在时为14.7μmol·L-1。结论表达的m2-Gilα融合蛋白具备M2配体结合的特性及G蛋白耦联受体的信号转导功能,对于有基础活性的m2-Gilα融合蛋白,Ach, Pilo是完全激动剂;atropine,alcuronium。
OBJECTIVE To identify the speeific ligands for M2 using m2-Gilα fusion protein by detecting the signaling transduction of its two partners.METHODS m2-Gilα fused cDNAs was generated via a two-step PCR protocol and then expressed with baculovirus-Sf9 cell system. [3H]QNB saturation binding in Sf9 membrane was performed to determine the expression level of m2 protein or m2-Gilα fusion protein. High affinity [35S] GTPγS binding detected the effect of various M2 ligands on [35S] GTPγS binding in the coexistence of GDP, GTP or ATP.RESULTS The expression level of m2-Gilα was (18.14 ±0.17) pmol·mg(pro)-1 .In the presence of 10 μmol·L-1GTPγS, [3H]QNB binding with m2-Gilα fusion protein decreased while [3H]QNB binding with m2 protein was not influenced.The curves shifted right on the existence of muscarinic agonists comparing with their absence,and the left shift was observed with muscarinic antagonist. IC50 values of GDP for Ach.Pilo, ligand-free, alcuronium, AF-DX116, Iso and atropine were 168.4,152.3,14.7,6.84,6.12,5.59 and 4.52 μmol·L-1 respectively. CONCLUSION m2-Cilα fusion protein has the pharmacological specificity of muscarinic receptor and the efficient signal of the two partners. The affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands. Ach and Pilo are agonists for m2-Gilα fusion protein, but atropine, alcuronium, AF-DX116 and Iso are antagonists or superantagonists.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2005年第11期868-872,共5页
Chinese Pharmaceutical Journal
基金
国家自然科学基金资助项目(30171077)辽宁省自然科学基金项目(002039)
