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

doi:10.3969/j.issn.1006-7795.2018.06.020

Abstract

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

CLC Number:

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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URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2018.06.020

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2018/V39/I6/905

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