Quantifying the diffusion of the agonist-occupied adenosine-A3 receptor at the single cell level

S.J. Briddon & S.J. Hill. Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, NG7 2UH.

We have previously shown that ABEA-X-BY630, a fluorescent derivative of N-ethyl carboxamidoadenosine (NECA) is an agonist at both adenosine-A1 and –A3 receptors (A1-AR and A3-AR respectively; Briddon et al., 2004a; Cordeaux et al., 2004). We have also demonstrated that binding of this agonist to the A1-AR can be monitored in unspecified microdomains of living cells using fluorescence correlation spectroscopy (FCS) (Briddon et al., 2004a). Here we have used ABEA-X-BY630 in combination with FCS to quantify the diffusion characteristics of the agonist-occupied human A3-AR.

Chinese Hamster Ovary (CHO) cells expressing the human adenosine-A3 receptor (CHO-A3 cells; Cordeaux et al., 2004) were seeded onto Nunc Labtek 8-well glass dishes and FCS experiments were performed in HEPES-buffered saline solution (HBSS) at 22±2°C, as previously reported (Briddon et al., 2004b). Autocorrelation analysis was performed using Zeiss AIM software as previously described (Briddon et al., 2004b). Data are quoted as mean ± s.e.mean, with ‘n’ indicating the number of cells on which measurements were performed. Statistical analysis was by unpaired Student’s t-test.

Initial FCS measurements of ABEA-X-BY630 (50nM in HBSS) indicated that free ligand had a diffusion time of 64±1μs (n=4). Subsequent FCS measurements were performed on the upper membrane of single CHO-A3 cells following incubation with ABEA-X-BY630 (2.5nM, 10 min, 22ºC). Here, in addition to free ligand diffusion ( τD1 ), two slower-diffusing species were detected (τD2 = 5.9±0.7ms and τD3 = 131±15ms, n=49). Interestingly, in contrast to the A1-AR, τD3 represented a higher proportion (75%) of the total binding detected (total binding=20.0±3.1, τD2 =5.1±0.6 and τD3 =14.9±2.5 receptors/ μm 2, n=33). To confirm that these slower-diffusing species represented agonist-occupied A3-ARs, similar experiments were performed in cells pre-incubated with the A3-AR antagonist, MRS1220 (100nM, 37°C). Total binding was reduced to 5.8±0.8 receptors/ μm2 (n=27; P<0.001 vs. control). This consisted of reductions in both τD2 (to 2.6±0.4 receptors/ μm2, P<0.01) and τD3 (to 3.3±0.4 receptors/μm2, P<0.001). MRS1220 had no significant effect on the diffusion times obtained for τD2 and τD3 (6.4±0.8 and 117±26ms, respectively).

These data show that ABEA-X-BY630 can be used to specifically measure the diffusional characterisitics of agonist-occupied A3-ARs in the microdomains of living cells. They also provide evidence for the presence of A3-ARs in large slowly diffusing complexes or compartments within the cell membrane and to a lesser extent in smaller faster diffusing membrane complexes.

Cordeaux, Y. et al. (2004) Proceedings of the British Pharmacological Society at http://www.pA2online.org/Vol2Issue4abst112P.html
Briddon, S.J. et al. (2004a) Proceedings of the British Pharmacological Society at http://www.pA2online.org/Vol2Issue4abst029P.html
Briddon, S.J. et al. (2004b) Proc. Natl. Acad. Sci. (USA), 101, 4673-4678.

This work was supported by the BBSRC.