TY - JOUR
T1 - Quantitative analysis of the formation and diffusion of A 1-adenosine receptor-antagonist complexes in single living cells
AU - Briddon, S. J.
AU - Middleton, R. J.
AU - Cordeaux, Y.
AU - Flavin, F. M.
AU - Weinstein, J. A.
AU - George, M. W.
AU - Kellam, B.
AU - Hill, S. J.
PY - 2004/3/30
Y1 - 2004/3/30
N2 - 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 A 1-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 (τD) of 62 μs (τD1, free ligand), 17 ms (τD2, A1-AR-ligand), and 320 ms (τD3). Confirmation that τD2 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 τ D2 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.
AB - 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 A 1-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 (τD) of 62 μs (τD1, free ligand), 17 ms (τD2, A1-AR-ligand), and 320 ms (τD3). Confirmation that τD2 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 τ D2 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.
UR - http://www.scopus.com/inward/record.url?scp=1842532195&partnerID=8YFLogxK
U2 - 10.1073/pnas.0400420101
DO - 10.1073/pnas.0400420101
M3 - Article
C2 - 15070776
AN - SCOPUS:1842532195
SN - 0027-8424
VL - 101
SP - 4673
EP - 4678
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 13
ER -