BINDING OF [H-3] DIHYDROTETRABENAZINE AND [I-125] AZIDOIODOKETANSERIN PHOTOAFFINITY-LABELING OF THE MONOAMINE TRANSPORTER OF PLATELET 5-HT ORGANELLES

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage orgaoelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodoketanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 ±1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values -1.1 ±0.2, 1.2 ±0.1 and 0.52 ±0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine. © 1990.