Optimizing the Expression of Recombinant αβγ GABAA Receptors in HEK293 Cells for High-Throughput Screening

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS SLAS Discovery Pub Date : 2009-01-01 DOI:10.1177/1087057108328017

Daniel Gilbert , Abolghasem Esmaeili , Joseph W. Lynch

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Abstract

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABA_ARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABA_ARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers’ instructions, conferred the strongest and most reliable expression of trimeric αβγ GABA_ARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABA_ARs provides an effective means of discovering novel GABA_AR modulators for use as therapeutic lead compounds and pharmacological probes.

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重组αβγ GABAA受体在HEK293细胞高通量筛选中的表达优化

尽管是重要的临床靶点，但可靠表达重组三聚体αβγ GABA-A受体（GABAARs）进行高通量筛选并不简单。因此，本研究寻求一种简单可靠的方法在HEK293细胞中瞬时表达α1β1γ1和α1β1γ2 GABAARs。通过流式细胞术和基于阴离子敏感的黄色荧光蛋白的药理学分析，评估了5种不同转染策略的表达效率。PolyFect™和Effectene™，根据制造商的说明，给予最强和最可靠的三聚体αβγ GABAARs的表达。黄荧光蛋白功能分析显示，含γ - 1和γ - 2受体的药理特性存在显著差异，与之前的电生理表征一致。作者认为，这种表达和筛选重组GABAARs的方法为发现新的GABAAR调节剂作为治疗先导化合物和药理探针提供了有效的手段。

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来源期刊

SLAS Discovery Chemistry-Analytical Chemistry

CiteScore

7.00

自引率

3.20%

发文量

审稿时长

39 days

期刊介绍： Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).