Optimizing the expression of recombinant alphabetagamma GABAA receptors in HEK293 cells for high-throughput screening.

Q2 Chemistry

Journal of Biomolecular Screening Pub Date : 2009-01-01 DOI:10.1177/1087057108328017

Daniel Gilbert, Abolghasem Esmaeili, Joseph W Lynch

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引用次数: 17

Abstract

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric alphabetagamma GABA-A receptors (GABA(A)Rs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing alpha1beta1gamma1 and alpha1beta1gamma2 GABA(A)Rs 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 alphabetagamma GABA(A)Rs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of gamma1- and gamma2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABA(A)Rs provides an effective means of discovering novel GABA(A)R modulators for use as therapeutic lead compounds and pharmacological probes.

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优化重组alphabetagamma GABAA受体在HEK293细胞中的表达，进行高通量筛选。

尽管是重要的临床靶点，但可靠表达重组三聚体GABA-A受体(GABA(A)Rs)进行高通量筛选并不简单。因此，本研究试图设计一种简单可靠的方法，在HEK293细胞中瞬时表达alpha1beta1gamma1和alpha1beta1gamma2 GABA(a)Rs。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，根据制造商的说明使用，赋予三聚体字母表GABA(A)Rs最强和最可靠的表达。通过黄色荧光蛋白检测的功能分析显示，含gamma1-和含gamma2受体的药理学特性存在显著差异，与之前的电生理表征一致。作者认为，这种表达和筛选重组GABA(A)Rs的方法为发现新的GABA(A)R调节剂作为治疗先导化合物和药理学探针提供了有效的手段。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Biomolecular Screening 生物-分析化学

CiteScore

2.41

自引率

0.00%

发文量

审稿时长

4-8 weeks

期刊介绍： Advancing the Science of Drug Discovery: 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.