Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.
Talwar S, Lynch J, Gilbert D (2013)
Publication Status: Published
Publication Type: Journal article, Original article
Publication year: 2013
Journal
Publisher: Public Library of Science
Book Volume: 8
Pages Range: e58479
Journal Issue: 3
DOI: 10.1371/journal.pone.0058479
Abstract
Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR) chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems-level analysis of ion channel properties in health and disease and the discovery of therapeutics to reverse pathological alterations.
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APA:
Talwar, S., Lynch, J., & Gilbert, D. (2013). Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells. PLoS ONE, 8(3), e58479. https://doi.org/10.1371/journal.pone.0058479
MLA:
Talwar, S, JW Lynch, and Daniel Gilbert. "Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells." PLoS ONE 8.3 (2013): e58479.
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