烟碱型乙酰胆碱受体β4亚基点突变体的构建

Construction of Nicotinic Acethlcholine Receptor β4 Subunit Mutant

利用PCR介导的定点突变技术构建包含β4亚基的烟碱型乙酰胆碱受体点突变体模型,同时采用双电极电压钳技术,对激动剂ACh和拮抗剂α-CTx RegIIA对突变受体的活性进行检测。结果表明:β4亚基胞外N端序列第168位的异亮氨酸(Ile)成功突变成了丝氨酸(Ser),突变后的受体对激动剂ACh的活性降低,对拮抗剂α-CTx RegIIA的活性降低了1.4倍,半阻断剂量(IC50)为170 nmol·L^-1。

To explain the molecular mechanisms of interaction between β4 nAChR subunit and ligand(agonist ACh and inhibitor α-CTx RegIIA), a mutant of β4 nAChR subunit was constructed by substituting 168 th Ile(I) with Ser(S) in the Loop F of N-terminal ligand-binding region, by using the method of site-directed mutagenesis that PCR mediated. The results showed that the inward current of oocytes injected with cRNA of α3β4[S168 I] nAChR could be recorded by two-electrode voltage clamp. The mutant receptor had 1.4 times lower sensitivity to α-CTx RegIIA with a higher IC50 of 170 nmol·L^-1, compared with the wild-type α3β4 nAChR.