Quantitative analysis of the formation and diffusion of A ₁-adenosine receptor-antagonist complexes in single living cells

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 (pK_b) = 6.7)] and stimulation of inositol phosphate accumulation (pK_b = 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 (τ_D) of 62 μs (τ_D1, free ligand), 17 ms (τ_D2, A₁-AR-ligand), and 320 ms (τ_D3). Confirmation that τ_D2 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.