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Plos One : Yellow Fluorescent Protein-based Assay to Measure Gabaa Channel Activation and Allosteric Modulation in Cho-k1 Cells, Volume 8

By Rudolph, Uwe

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Book Id: WPLBN0003969643
Format Type: PDF eBook :
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Reproduction Date: 2015

Title: Plos One : Yellow Fluorescent Protein-based Assay to Measure Gabaa Channel Activation and Allosteric Modulation in Cho-k1 Cells, Volume 8  
Author: Rudolph, Uwe
Volume: Volume 8
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection (Contemporary)
Historic
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Publisher: Plos

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Rudolph, U. (n.d.). Plos One : Yellow Fluorescent Protein-based Assay to Measure Gabaa Channel Activation and Allosteric Modulation in Cho-k1 Cells, Volume 8. Retrieved from http://netlibrary.net/


Description
Description : The c-aminobutyric acid A (GABAA) ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABAA channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABAA ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP)-based assay to be able to study allosteric modulation of the GABAA ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABAA a2b3c2 along with a halide sensing YFP-H148Q/I152L (YFP-GABAA2 cells). As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABAA subunit composition and functionality. We found that the I2 concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS) and could be used to discover novel modulators acting on GABAA ion channels.

 

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