title: Aequorin functional assay for characterization of G-protein-coupled receptors: implementation with cryopreserved transiently transfected cells.
abstract: Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M(1) receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.
authors.affiliation: Cephalon, Inc., Worldwide Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380, USA. bjones@cephalon.com
authors.forename: Bruce
authors.initials: B
authors.lastname: Jones
authors.forename: Beverly
authors.initials: B
authors.lastname: Holskin
authors.forename: Sheryl
authors.initials: S
authors.lastname: Meyer
authors.forename: Thao
authors.initials: T
authors.lastname: Ung
authors.forename: Vincent
authors.initials: V
authors.lastname: Dupriez
authors.forename: Sandra Y
authors.initials: SY
authors.lastname: Flores
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Burgeon
authors.forename: Mark
authors.initials: M
authors.lastname: Ator
authors.forename: Emir
authors.initials: E
authors.lastname: Duzic
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000331:Aequorin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015925:Cryopreservation; D004072:Digitonin / Q000378:metabolism; D006801:Humans; D010095:Oxotremorine / Q000378:metabolism; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; D000331:Aequorin; D010095:Oxotremorine; D004072:Digitonin; D000109:Acetylcholine; D002118:Calcium