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.

Fig. 1.

The chemical structure of XAC (a) and XAC-BY630 (b).

Fig. 2.

Visualization of the A₁-AR in live CHO-A1 cells. CHO-A1 cells were incubated at 22°C with 10-250 nM XAC-BY630 for 15 min (a), 50 nM XAC-BY630 for 0-30 min (b), or DPCPX (1-20 nM, 30 min, 37°C) (c) followed by 50 nM XAC-BY630 for 15 min before capture of single confocal images. (c Right) Native CHO-K1 cells were incubated with XAC-BY630 (100 nM,15 min). Each experiment shown is representative of four performed.

Fig. 3.

Visualization of XAC-BY630 binding to CHO-A₁Tpz cells. Cells were incubated with XAC-BY630 (100 nM, 30 min, 22°C), and simultaneous confocal images were captured with 488-nm excitation (a,A₁-ARTpz) and 633-nm excitation (b, XAC-BY630). The overlay image (c) represents colocalized pixels as yellow/orange. Colocalization analysis showed those pixels with the highest degree of colocalization (d). The image shown is representative of five similar experiments performed.

Fig. 4.

FCS analysis of A₁-AR-Tpz expressed in CHO cells. FCS measurements were performed in CHO-A1Tpz cells at 22°C as described. (a) The confocal volume was positioned over the cell nucleus in x-y, and a z-intensity scan was performed. Peaks in intensity corresponded to the lower membrane (LM) and upper cell membranes (UM). The vertical line represents the measurement position. (b) Intensity fluctuations (Upper) were collected for 30 s, producing the normalized autocorrelation curve shown (Lower). Data were best fit by a two-component 2D model, which, in this instance, gave τ_D1 = 45 μs [fraction (f₁) = 0.46], τ_D2 = 12.4 ms (f₂ = 0.54).

Fig. 5.

Diffusional analysis of XAC-BY630 binding to CHO-A1 cells by using FCS. CHO-A1 cells were incubated with XAC-BY630 (2.5 nM, 15 min). (a) After a z-intensity scan (Right). FCS readings were taken at three different z-positions within the same cell; A, extracellular; B, upper membrane; and C, intracellular. (b) Intensity fluctuations (Upper) were recorded for 30 s, after a 15-s prebleach, and subsequent normalized autocorrelation analyses are shown (Lower). Position A, τ_D1 = 65 μs(f₁ = 1); position B, τ_D1 = 65 μs(f₁ = 0.50), τ_D2 = 18.4 ms (f₂ = 0.27), τ_D3 = 264 ms (f₃ = 0.23); position C, τ_D1 = 7.3 ms (f₁ = 1).

Fig. 6.

XAC-BY630 binding to the membrane of single CHO-A1 cells quantified by using FCS. (a) CHO-A1 cells were incubated with XAC-BY630 (2.5 nM, 30 min) and the confocal volume positioned either (i) in the extracellular buffer or (ii) on the upper membrane. Intensity fluctuations were recorded (Upper), and the subsequent normalized autocorrelation curves are shown (Lower). Diffusion components were assigned as follows: (i) τ_D1 = 64 μs(f₁ = 1), (ii) τ_D1 = 64 μs(f₁ = 0.24), τ_D2 = 12.5 ms (f₂ = 0.65), τ_D3 = 300 ms (f₃ = 0.11). (b) Measurements as described in a were performed on cells incubated with 1-40 nM XAC-BY630 (30 min, 22°C). For each cell, the free ligand (τ_D1) and corresponding amount of receptor-ligand complex (τ_D2) were calculated. The plot of [Free] vs. [Bound] is shown (closed circles). Each point is representative of measurements taken on 7-16 cells, in four to eight experiments. The data have been fitted to a single component binding isotherm (solid line), which yields B_max = 75 nM, and K_d = 33 nM. A further set of experiments were performed in CHO-A1 cells exposed to DPCPX (1 μM, 30 min) before addition of XAC-BY630 (open circles). Each point is representative of measurements taken on 6-12 cells, within three to six independent experiments.