α7烟碱型乙酰胆碱受体点突变体的制备及其功能研究

Preparation and functional study of point mutant of α7 nicotinic acetylcholine receptor

目的利用氨基酸定点突变技术以大鼠野生型α7烟碱型乙酰胆碱受体(nAChR)为模板制备α7nAChR突变体。方法采用体外转录、定点突变、凝胶电泳、双电极电压钳等技术对α7nAChR进行点突变,在非洲爪蟾卵母细胞上表达α7nAChR及其突变体,并研究其受体活性功能。结果将α7nAChR第111位的丙氨酸突变为丝氨酸,制备了α7nAChR的点突变体,测定了突变体对激动剂乙酰胆碱(ACh)的半数效应浓度(EC_(50))为165.6μmol/L。结论该结果不仅为受体定点突变提供了1种方法,同时为药物筛选和研究α7nAChR结构与功能关系提供了模型。

Objective To construct the point mutant of ratα7nicotinic acetylcholine receptor（nAChR）and evaluate its function.Methods Site-directed mutagenesis was used to construct α7 nAChR point mutant,which was expressed in Xenopus oocytes and functional tested by electrophysiology with two-electrode voltage-clamp techniques.Results An α7 nAChR point mutant was constructed by mutating Alanine（Ala）at position 111of α7 nAChR to Serine（Ser）successfully.The EC（50） of the α7 nAChR mutant to the agonist acetylcholine（ACh）was 165.6μmol/L.Conclusion The results would provide an effective method for site-directed mutagenesis,and offer a realistic model for drug screening and characterization of structure-function relationships of α7 nAChR.