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

Kruger, Wade A.; Gilbert, Daniel F.; Hawthorne, Rebecca; Hryciw, Deanne H.; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W.

doi:10.1016/j.neulet.2005.01.065

Cited by 56 publications

(58 citation statements)

References 26 publications

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“…Functional High Throughput Analysis-The first round of functional characterization involved imaging live transfected cells via an automated fluorescence-based screening system using YFP-I152L fluorescence quench as an indicator of anion influx rate (20). The advantage of this approach over electro- physiology is that responses of large cell numbers can be averaged, thus permitting the reliable quantitation of small changes in the functional expression levels of mutated GlyR isoforms.…”

Section: Resultsmentioning

confidence: 99%

“…Within the following 24 -32 h, the cell culture medium was replaced by an extracellular control solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose, pH 7.4). Cells were imaged with an automated fluorescence-based screening system using YFP-I152L fluorescence quench as an indicator of anion influx rate (20). During experiments, fluorescence images of each well were obtained twice: once before and once after the application of a sodium iodide solution (140 mM NaI, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose, pH 7.4) containing defined concentrations of glycine.…”

Section: Methodsmentioning

confidence: 99%

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New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

Bode

Wood

Mullins

et al. 2013

Journal of Biological Chemistry

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Background: Hyperekplexia mutations have provided much information about glycine receptor structure and function. Results: We identified and characterized nine new mutations. Dominant mutations resulted in spontaneous activation, whereas recessive mutations precluded surface expression. Conclusion: These data provide insight into glycine receptor activation mechanisms and surface expression determinants. Significance: The results enhance our understanding of hyperekplexia pathology and glycine receptor structure-function.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

Bode

Wood

Mullins

et al. 2013

Journal of Biological Chemistry

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“…Cells were imaged using an automated high-content screening system using YFP-I152L fluorescence as an indicator of Cl Ϫ influx rate (Kruger et al, 2005;Gilbert et al, 2009). Briefly, HEK293 cells were cotransfected with mutant or WT pRK5-hGlyR␣1 and pcDNA3.1-YFP-I152L and plated into a 384-well plate (ϳ2.5 ϫ 10 3 cells/well).…”

Section: Methodsmentioning

confidence: 99%

Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

Chung¹,

Vanbellinghen²,

Mullins³

et al. 2010

Journal of Neuroscience

116

152

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Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) ␣1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric ␣1 or heteromeric ␣1␤ GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

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“…GlyR activation was quantitated by monitoring the fluorescence emission intensity of YFP-I152L, which is potently quenched by iodide ions (11). These anions permeate efficiently into the cell through activated GlyRs (12). We have previously shown that this YFP-I152L fluorescence quench is potently antagonized by the GlyR-specific antagonists, picrotoxin and strychnine (12).…”

Section: Molecular Constructsmentioning

confidence: 99%

“…The later project involves screening a large random mutant library for mutant clones that alter GlyR pharmacological properties. Because GlyR activation gates a Cl 2 influx, its activation can be monitored using yellow fluorescent protein (YFP) variants that are highly sensitive to quenching by small anions and are thus suited to reporting anionic influx into cells (11,12). Currently, fluorescence quenching of adherent cells in response to agonist is assessed using a specialized imaging microscope plate-based system.…”

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confidence: 99%

Multiplexed labeling of viable cells for high‐throughput analysis of glycine receptor function using flow cytometry

et al. 2009

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Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. ' 2009 International Society for Advancement of Cytometry Key terms multiplex; chloride channels; glycine receptor FLOW cytometry permits multiple cell parameters to be analyzed simultaneously with high sensitivity and a high degree of statistical robustness. It is emerging as an important drug discovery tool because it can screen test compounds against a wide variety of cellular targets simultaneously. However, achieving this goal is dependant on two factors: (1) the ability to automate the screening of large numbers of compounds, and (2) the ability to monitor multiple parameters simultaneously in live cells. Recent developments (reviewed by Sklar et al. (1)), while useful, require dedicated hardware on an unmodified flow cytometer.Multiplexed or duplexed analysis allows the screening of multiple discrete samples in one tube or well at the same time (2-4). A recent method (5), which introduced the idea of labeling discrete cell populations with a unique combination of fluorochromes, demonstrated the feasibility of employing flow cytometry to discriminate between different multiplexed labeled cell populations. However, this method required cellular fixation, preventing further culture or experimentation.Glycine receptors (GlyRs) are pentameric Cys-loop anion-permeant channels that mediate inhibitory neurotransmission in the spinal cord, brainstem, and retina (6,7). These receptors have recently emerged as therapeutic targets for chronic inflammatory pain and movement disorders (8-10). Our interest lies in discovering novel bioactive compounds with therapeutic potential and i...

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

Cited by 56 publications

References 26 publications

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

Multiplexed labeling of viable cells for high‐throughput analysis of glycine receptor function using flow cytometry

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