Interaction of the mioflazine derivative R75231 with the nucleoside transporter: evidence for positive cooperativity

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [3H]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a 'mixed' type inhibitor of [3H]nitrobenzylthioinosine binding (increased KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax of 44 pmol/mg protein, which is comparable to the number of [3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [3H]R75231 and actually caused an initial increase in the amount of site-bound [3H]R75231. Adenosine, on the other hand, enhanced the rate of [3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.