title: Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells.
abstract: The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros (tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3). Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A1-AR-Topaz fusion protein (15 ms). Quantification of tauD2 showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A1-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A1-AR interactions in single living cells in health and disease.
authors.affiliation: Institute of Cell Signalling, Medical School, University of Nottingham, Nottingham NG7 2UH, United Kingdom.
authors.forename: S J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: R J
authors.initials: RJ
authors.lastname: Middleton
authors.forename: Y
authors.initials: Y
authors.lastname: Cordeaux
authors.forename: F M
authors.initials: FM
authors.lastname: Flavin
authors.forename: J A
authors.initials: JA
authors.lastname: Weinstein
authors.forename: M W
authors.initials: MW
authors.lastname: George
authors.forename: B
authors.initials: B
authors.lastname: Kellam
authors.forename: S J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D058916:Adenosine A1 Receptor Antagonists; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000502:physiology; D006224:Cricetinae; D017931:DNA Primers; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D043682:Receptor, Adenosine A1 / Q000502:physiology*; D014970:Xanthines / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D058916:Adenosine A1 Receptor Antagonists; D017931:DNA Primers; D043682:Receptor, Adenosine A1; D014970:Xanthines; C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine