A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

Kruger, Wade, Gilbert, Daniel, Hawthorne, Rebecca, Hryciw, Deanne H., Frings, Stephan, Poronnik, Philip and Lynch, Joseph W. (2005) A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels. Neuroscience Letters, 380 3: 340-345.


Author Kruger, Wade
Gilbert, Daniel
Hawthorne, Rebecca
Hryciw, Deanne H.
Frings, Stephan
Poronnik, Philip
Lynch, Joseph W.
Title A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels
Journal name Neuroscience Letters  (ERA 2012 Listed)    (ERA 2010 Rank C)   Check publisher's open access policy
Publication date 2005-06-03
Sub-type Article
DOI 10.1016/j.neulet.2005.01.065
Volume number 380
Issue number 3
ISSN 0304-3940
Start page 340
End page 345
Total pages 6
Editor S. G. Waxman
Place of publication Clare
Publisher Elsevier Ireland Ltd
Collection year 2005
Language eng
Subject C1
270601 Animal Physiology - Biophysics
730104 Nervous system and disorders
Abstract There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
Keyword Ligand-gated Ion Channel
Yfp
Gfp
Green Fluorescent Protein
Drug Discovery
Neurosciences
Gene Family
Subunit
Identification
Sensitivity
Variants
Neurons
Binding
Domain
Q-Index Code C1

 
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