The serotonin transporter from human brain: Purification and partial characterization

The serotonin (5-HT) transporter from human striatum was solubilized by digitonin and purified by affinity chromatography. The native protein-detergent complex had a molecular mass of 205 kDa, as estimated by gel-exclusion chromatography of the eluates obtained From affinity chromatography. 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, and 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 preparations. [H-3]-imipramine labeled a high- and a low-affinity site on parent membranes, whereas it bound to a single high-affinity site on the purified extract. Tricyclic antidepressants, selective serotonin reuptake inhibitors and 5-HT itself displaced [H-3]-paroxetine and [H-3]-imipramine from their high-affinity binding sites on both the membrane-bound 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 identical protein of 205 kDa. Our results suggest overlapping binding sites for tricyclic antidepressants, selective serotonin reuptake inhibitors and 5-HT on the 5-HT transporter.