Establishment of Human GABA<sub>A</sub>R-CHO Cell Line Stable Expression

doi:10.13523/j.cb.2108063

China Biotechnology

2022, Vol. 42

Issue (3): 38-46 DOI: 10.13523/j.cb.2108063

Establishment of Human GABA_AR-CHO Cell Line Stable Expression

ZHANG Yi,WANG Chen,SHI Jing-jing,CHEN Xue-jun,ZHANG Rui-hua,JIN Qian,SHI Tong^*(

),LI Li-qin^*(

)

State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China

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Abstract

Objective: A functional α₁β₂γ_2L-GABA_AR-CHO human cell line is constructed in which α₁ subunit is inducd expression and β₂ and γ_2L subunits are stable expression. Methods: The coding genes of human subunit α₁, β₂ and γ_2L were amplified from human cDNA library, and the subunit vectors were respectively constructed. The three subunit vectors were cotransfected into CHO-K1 cells, and the stable expression clones were screened by resistance screening and membrane potential detection. The expression of subunits were identified by qPCR and western blot;the pharmacological function of α₁β₂γ_2L-GABA_AR-CHO line were identified by whole-cell patch clamp detection and membrane potential detection method. Results: The α₁β₂γ_2L-GABA_AR-CHO with high expression level was obtained by screening the clones. The cells stably expressed α₁, β₂ and γ_2L subunits. The constructed α₁β₂γ_2L-GABA_AR-CHO cells expressed α₁ subunit in the presence of tetracycline, and assembled with β₂ and γ_2L subunits to form α₁β₂γ_2L-GABA_AR with functional activity. Whole-cell patch clamp detection of α₁β₂γ_2L-GABA_AR-CHO showed that GABA could stimulate it and cause the characteristic current change of chloride channel in α₁β₂γ_2L-GABA_AR-CHO, and diazepam could enhance the activation effect of GABA on α₁β₂γ_2L-GABA_AR. Membrane potential detection showed that EC₅₀ of agonist GABA was (177.72±15.92) nmol/L, EC₅₀ of allosteric agent diazepam was (3.63±0.52) μmol/L, and IC₅₀ of antagonist bicuculine was (538.83±29.55) nmol/L, respectively. Conclusion: α₁β₂γ_2L-GABA_AR-CHO cell line is successfully constructed by the induced expression strategy, which has the pharmacological function of specific detection of agonists, positive allosteric agents and antagonists.

Key words： α₁β₂γ_2L-GABA_AR-CHO Induced expression Membrane potential detection GABA Diazepam

Received: 27 August 2021 Published: 07 April 2022

ZTFLH:

Q819

Corresponding Authors: Tong SHI,Li-qin LI E-mail: tong198282@126.com;llq969696@163.com

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	ZHANG Yi
	WANG Chen
	SHI Jing-jing
	CHEN Xue-jun
	ZHANG Rui-hua
	JIN Qian
	SHI Tong
	LI Li-qin

Cite this article:

ZHANG Yi, WANG Chen, SHI Jing-jing, CHEN Xue-jun, ZHANG Rui-hua, JIN Qian, SHI Tong, LI Li-qin. Establishment of Human GABA_AR-CHO Cell Line Stable Expression. China Biotechnology, 2022, 42(3): 38-46.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2108063 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I3/38

Table 1 qPCR primers for α₁, β₂, γ_2L, GAPDH

Fig.1 Expression vector map of GABA_AR subunits (a) pcDNA4/TO/zeo-α₁ (b) pIRES/puro3-β₂ (c) pcDNA3.1/hygro-γ_2L

Fig.2 Expression of α₁β₂γ_2L-GABA_AR detected by FLIPR membrane potential

Fig.3 Expression of α₁, β₂ and γ_2L subunits in GABA_AR cell line identified by qPCR (a) GABA_AR-CHO-α₁, n=3 (b) GABA_AR-CHO-β₂, n=3 (c) GABA_AR-CHO-γ_2L, n=3. ^* P<0.05, ^** P< 0.01

Fig.4 Expression of α₁ and γ_2L subunits in GABA_AR cell line identified by western blot (a) Bands of protein expression (b) Relative expression of α₁ subunit protein, n=3 (c) Relative expression of γ_2L subunit, n=3.^*p<0.05,^** P<0.01

Fig.5 The activation effects of tool drugs GABA and Dia detected by whole cell patch clamp

Fig.6 Dose-effect relationship of GABA, Dia and bicuculine detected by membrane potential (a) Dose-effect relationship of GABA (b) Dose-effect relationship of Dia (c) Dose-effect relationship of bicuculine


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