利用双电极电压钳检测rs12899798单核苷酸多态性对α7烟碱受体功能的影响

doi:10.3969/j.issn.1006-7795.2018.06.020

首都医科大学学报 ›› 2018, Vol. 39 ›› Issue (6): 905-909.doi: 10.3969/j.issn.1006-7795.2018.06.020

利用双电极电压钳检测rs12899798单核苷酸多态性对α7烟碱受体功能的影响

薛奋勤, 薛冰

首都医科大学中心实验室, 北京 100069

收稿日期:2018-06-12 出版日期:2018-11-21 发布日期:2018-12-19
通讯作者: 薛冰 E-mail:xuebing_bj@126.com

Analysis of the functional impact of the rs12899798 single nucleotide polymorphism in human α7 nicotinic receptor by two-electrode voltage-clamp

Xue Fenqin, Xue Bing

Core Facilities Center, Capital Medical University, Beijing 100069, China

Received:2018-06-12 Online:2018-11-21 Published:2018-12-19

摘要/Abstract

摘要： 目的检测α7烟碱受体（α7 nicotinic receptor，nAChR）的一个错义突变（W55G）对受体功能的影响。方法利用双电极电压钳在非洲爪蟾卵母细胞检测了突变体W55G的功能。首先将nAChR α7亚基的cDNA克隆进入pGEMHE载体中，利用定点突变的方法构建突变体W55G。然后在体外利用T7 RNA聚合酶合成cRNA，并利用显微注射的方法将cRNA注射进入非洲爪蟾卵母细胞中。1~3 d后，利用双电极电压钳检测野生型nAChR α7及其突变体W55G的功能。结果 W55G突变体对乙酰胆碱敏感性降低约6倍，但最大电流（I_max）则显著增加；对胆碱的敏感性降低了约3倍。此外，W55G突变体明显比野生nAChR有更高的时间常数，明显降低了α7烟碱受体的脱敏速率。结论 nAChR α7 突变体功能的改变可能重塑突触后反应，对于脑疾病的发生可能会有较大影响。

关键词: α7烟碱受体, 双电极电压钳, 单核苷酸多态性

Abstract: Objective To test the impact of a miss-sense mutation (W55G) on the α7 nicotinic receptor nAChR(α7) function. Methods The receptor function in Xenopus oocytes was tested with two-electrode voltage-clamp. The cDNAs encoding human wild type nAChR α7 subunit was cloned into pGEMHE vector with T7 orientation. W55G mutation was made with the PCR-based quick change method of the site-directed mutagenesis. The cRNAs were transcribed by standard in vitro transcription using T7 RNA polymerase, and then they were injected into oocytes with a Nanoject micro-injection system. 1 to 3 days after injection, the oocytes expressing wild type or the mutant receptor were voltage-clamped at -60 mV to measure agonist-induced currents using a GeneClamp 500B amplifier. Results Our results revealed that W55G mutant exhibited a-6 folds decrease of the acetylcholine sensitivity but with several fold increase in the maximal current. Furthermore, the W55G showed a-3 folds reduction of the choline sensitivity. In addition, the W55G mutant showed significantly larger time constants, suggesting the slower current desensitization rate. Conclusion The reduced sensitivity of the receptor to its neurotransmitter and slowed desensitization rate might reshape the postsynaptic response, implying potential impact on nAChR α7-mediated brain disorder.

Key words: α7 nicotinic receptor, two-electrode voltage-clamp, single nucleotide polymorphism

中图分类号:

薛奋勤, 薛冰. 利用双电极电压钳检测rs12899798单核苷酸多态性对α7烟碱受体功能的影响[J]. 首都医科大学学报, 2018, 39(6): 905-909.

Xue Fenqin, Xue Bing. Analysis of the functional impact of the rs12899798 single nucleotide polymorphism in human α7 nicotinic receptor by two-electrode voltage-clamp[J]. Journal of Capital Medical University, 2018, 39(6): 905-909.

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利用双电极电压钳检测rs12899798单核苷酸多态性对α7烟碱受体功能的影响

Analysis of the functional impact of the rs12899798 single nucleotide polymorphism in human α7 nicotinic receptor by two-electrode voltage-clamp

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