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

目的检测α7烟碱受体(α7 nicotinic receptor,n AChR)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n AChRα7亚基的c DNA克隆进入p GEMHE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n AChRα7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(Imax)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n AChR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 n AChRα7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。

Objective To test the impact of a miss-sense mutation(W55G)on theα7nicotinic receptor nAChR(α7)function.Methods The receptor function in Xenopus oocytes was tested with two-electrode voltage-clamp.The cDNAs encoding human wild type nAChRα7subunit was cloned into pGEMHE vector with T7orientation.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 T7RNA polymerase,and then they were injected into oocytes with a Nanoject micro-injection system.1to3days after injection,the oocytes expressing wild type or the mutant receptor were voltage-clamped at-60mV to measure agonist-induced currents using a GeneClamp500B amplifier.Results Our results revealed that W55G mutant exhibited a-6folds decrease of the acetylcholine sensitivity but with several fold increase in the maximal current.Furthermore,the W55G showed a-3folds 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 nAChRα7-mediated brain disorder.