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nAChRs α3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(EC50)分别为55.33和163.00 μmol·L-1,对突变型α3(S147T)β2 nAChR、α3(S147T)β4 nAChR的EC50分别为33.10和121.10 μmol·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。
Abstract
OBJECTIVE To construct the point mutants of α3* nicotine acetylcholine receptors (nAChRs), optimize the method of receptor mutagenesis and investigate the function of the mutants by using the agonist acetycholine (Ach). METHODS The α3* nAChRs mutants were constructed by PCR mediated site-directed mutation techniques. Point mutated primers were designed according to rat α3 subunit gene. The cRNA of α3 subunit point mutant was synthesized by in vitro transcription. The expression of mutants in Xenopus oocytes were detected by two-electrode voltage-clamp techniques. Gating properties of the two mutants were detected by Ach. RESULTS Mutants of α3β2 and α3β4 nAChRs subtypes were constructed successfully. The half effective concentrations (EC50) of wild types α3β2 and α3β4 nAChRs were 55.33 and 163.00 μmol·L-1, respectively. While the EC50 of α3(S147T)β2 and α3(S147T)β4 nAChRs mutants were 33.10 and 121.10 μmol·L-1, respectively. CONCLUSION The construction of mutation from the 147th serine to threonine of α3 subunit can provide a function model to make more other receptor mutants, and would be helpful to interrogate the interaction between drug and α3* nAChR.
关键词
烟碱型乙酰胆碱受体 /
α3亚基定点突变 /
双电极电压钳 /
受体功能
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Key words
nicotinic acetylcholine receptors /
α3 subunit site-directed mutation /
two-electrode voltage clamp /
receptor function
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金重点国际合作资助项目(81420108028);长江学者和创新团队发展计划资助项目(IRT_15R15);海南省普通高等学校研究生创新科研课题资助项目(Hys 2016-15)
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