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目的构建包含α3亚基的烟碱型乙酰胆碱受体(nicotinic acetylcholine receptors,nAChRs)点突变型,优化受体突变的方法,并利用nAChRs的激动剂乙酰胆碱(acetycholine,ACh)对突变型的功能进行研究。方法利用聚合酶链反应(PCR)介导的定点突变技术,设计引物并以大鼠nAChRsα3亚基的基因作为模版,获得α3亚基基因的点突变体,通过体外转录获得α3亚基突变体的cRNA。利用非洲爪蟾卵母细胞表达系统和双电极电压钳技术,对含有α3亚基点突变的2种受体亚型的表达情况进行检测。同时利用ACh对受体突变型的门控特性进行检测。结果成功建立了α3亚基第147位丝氨酸(serine,Ser,S)的突变体模型,激动剂ACh对野生型α3β2 nAChR和α3β4 nAChR的半数有效浓度(EC_(50))分别为55.33和163.00μmol·L~(-1),对突变型α3(S147T)β2 nAChR、α3(S147T)β4 nAChR的EC50分别为33.10和121.10μmol·L~(-1)。结论将α3亚基第147位Ser突变成苏氨酸(threonine,Thr,T)后,成功建立了2个具有功能型的突变体。α3亚基点突变受体模型的成功建立,可为研究药物与α3*nAChR相互作用的分子机制提供功能模型和研究基础,也可为更多受体突变型的建立提供方法借鉴。
OBJECTIVE: To construct the point mutation of nicotinic acetylcholine receptors (nAChRs) containing α3 subunits and to optimize the method of receptor mutation. The function of the mutant was studied by using acetycholine (ACh), an agonist of nAChRs, . Methods The polymerase chain reaction (PCR) -based site-directed mutagenesis was used to design the primers. The nAChRs α3 subunit gene was used as a template to obtain point mutants of α3 subunit gene. The α3 subunit mutants were obtained by in vitro transcription cRNA. Using Xenopus oocytes expression system and two-electrode voltage-clamp technique, the expression of two receptor subtypes containing point mutation of α3 subunit was detected. At the same time, ACh was used to detect the gating characteristics of receptor mutant. Results The mutant serine (Ser, S) mutant was successfully established. The half effective concentration (AC 50) of ACh against wild-type α3β2 nAChR and α3β4 nAChR was 55.33 and 163.00 μmol · L -1, the EC50 of mutant α3 (S147T) β2 nAChR and α3 (S147T) β4 nAChR were 33.10 and 121.10 μmol·L -1, respectively. Conclusion Two mutations with serine function were successfully constructed after threonine (Thr, T) was substituted into Ser at position 147 of α3 subunit. The successful establishment of the α3 subunit point mutation receptor model can provide a functional model and a research basis for studying the molecular mechanism of drug interaction with α3 * nAChR, and provide a reference for the establishment of more receptor mutants.