Live Cell Imaging Of The Kinetics Of Ligand Binding At The Human Adenosine A3 Receptor

Abstract

The aim of this study was to investigate the association and dissociation kinetics of fluorescently labelled adenosine ligands at the human adenosine A3 receptor at a single cell level. CHO cells stably expressing the human adenosine A3 receptor were exposed to 100 nM of fluorescent ligand after which cells were washed with buffer alone or in the presence of an unlabelled adenosine ligand. Confocal fluorescence and phase images were obtained using a Zeiss 510 confocal microscope.The association of ABA-X-BY630, a novel N6-aminoalkyl derivative of adenosine which incorporates the BODIPY [630/650] fluorophore, was monophasic with an association rate constant, kon, of 574700 ± 19000 M−1sec−1, n=7. ABA-X-BY630 dissociation was determined under conditions reflecting that of infinite dilution in the absence and presence of the selective adenosine A3 antagonist, MRS 1220. Under both conditions, ABA-X-BY630 dissociation was monophasic, however the dissociation rate in the absence of antagonist (koff = 0.019 ± 0.001 sec−1, n=4) was significantly slower than that in the presence of 1 μM MRS 1220 (koff = 0.080 ± 0.007 sec−1, n=4).In summary, confocal imaging has been used to directly measure, at single cell level, the binding kinetics of the fluorescent adenosine agonist, ABA-X-BY630. In addition, the perfusion system allows for the rapid removal of ligand and as such the comparison of ABA-X-BY630 dissociation in the absence and presence of antagonist. Under infinite dilution conditions, the dissociate rate of ABA-X-BY630 should be unaffected by the presence of a simple competitive antagonist. Therefore the ability of MRS 1220 to enhance the dissociation rate of ABA-X-BY630 suggests that there may be a negatively cooperative interaction occurring between the two ligands. Similar experiments have also been performed using additional fluorescently labelled adenosine ligands.