title: A cell-based functional assay using a green fluorescent protein-based calcium indicator dCys-GCaMP.
abstract: Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.
authors.affiliation: 1 Rugen Therapeutics Ltd. , Suzhou Industrial Park, China .
authors.forename: Bin
authors.initials: B
authors.lastname: Cai
authors.forename: Xia
authors.initials: X
authors.lastname: Chen
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Lijuan
authors.initials: L
authors.lastname: Gu
authors.forename: Jason R
authors.initials: JR
authors.lastname: Liu
authors.forename: Jay
authors.initials: J
authors.lastname: Liu
mesh_terms: D002118:Calcium / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003553:Cystine; D002118:Calcium
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