THE SEROTONIN TRANSPORTER FROM HUMAN BRAIN - PURIFICATION AND PARTIALCHARACTERIZATION

The serotonin (5-HT) transporter from human striatum was solubilized b y digitonin and purified by affinity chromatography. The native protei n-detergent complex had a molecular mass of 205 kDa, as estimated by g el-exclusion chromatography of the eluates obtained From affinity chro matography. The purified 5-HT transporter migrated as a single band of 67 kDa in SDS-PAGE. To clarify the spatial relationships between the binding sites of the tricyclic antidepressants, as [H-3]-imipramine, a nd of the selective serotonin reuptake inhibitors, as [H-3]-paroxetine , on the 5-HT transporter, both radioligands were used to label it in the purification steps. [H-3]-paroxetine bound with the same affinity to a single high-affinity site on both membrane and purified preparati ons. [H-3]-imipramine labeled a high- and a low-affinity site on paren t membranes, whereas it bound to a single high-affinity site on the pu rified extract. Tricyclic antidepressants, selective serotonin reuptak e inhibitors and 5-HT itself displaced [H-3]-paroxetine and [H-3]-imip ramine from their high-affinity binding sites on both the membrane-bou nd and the purified 5-HT transporter in a monophasic fashion with Hill coefficients close to unity. Furthermore, both [H-3]-paroxetine and [ H-3]-imipramine displayed a similar maximum binding capacity on an ide ntical protein of 205 kDa. Our results suggest overlapping binding sit es for tricyclic antidepressants, selective serotonin reuptake inhibit ors and 5-HT on the 5-HT transporter.