~ Pergamon 0197-01865(95)00080-1 Neurochem. Int. Vol. 28, No. 3, pp. 299-307, 1996 Copyright © 1996 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0197-0186/96 $15.00+0.00 THE SEROTONIN TRANSPORTER FROM HUMAN BRAIN : PURIFICATION AND PARTIAL CHARACTERIZATION A. ROTONDO, 1. G. GIANNACCINI, 2 L. BETTI, 2 G. CHIELLINI, ~ D. MARAZZITI, I C. MARTIN, 2 A. LUCACCHINI 2 and G. B. CASSANO I qnstitute of Psychiatry and 2Istituto Policattedra di Discipline Biologiche, University of Pisa, via Roma 67, 56100 Pisa, Italy (Received 14 February 1995 ; accepted 28 April 1995) Abstract--The serotonin (5-HT) transporter from hhman 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 [3H]-imipramine, and of the selective serotonin reuptake inhibitors, as [3H]-paroxetine, on the 5-HT transporter, both radioligands were used to label it in the purification steps. [3H]-paroxetine bound with the same affinity to a single high- affinity site on both membrane and purified preparations. [3H]-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 [3H]-paroxetine and [3H]-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 [3H]- paroxetine and [3H]-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. The synaptic level of serotonin (5-HT) is strictly regu- lated by an active transport mechanism that takes up the neurotransmitter from the synaptic cleft. A 5-HT re-uptake system has also been widely described in platelets, where it removes 5-HT from the blood stream for subsequent storage. The two transport sys- tems share remarkable similarities. Both of them require energy, are sodium- and temperature-depen- dent and are potently inhibited by tricyclic anti- depressants (TCAs) and by selective 5-HT reuptake inhibitors (SSRls) (Marcusson and Ross, 1990). A deeper knowledge of the structure/function of the 5-HT transporter might be helpful in clarifying the mode of action of TCAs and SSRIs and may shed some light on the pathophysiology of those psychiatric disorders, such as mood, anxiety and eating disorders, where these two classes of drugs are effective. * To whom all correspondence should be addressed at Lab- oratory of Neurogenetics, NIH/NIAAA, Flow Bldg, Room 2, 12501 Washington Avenue, Rockville MD 20852, U.S.A. Several efforts have been made to find out the molecular characteristics of the 5-HT transporter complex which has already been isolated using bio- chemical strategies from rat brain (Habert et al., 1986 ; Plenge et al., 1990, Graham et al., 1992) and human platelets (Rehavi et al., 1982; Rotman and Pribluda, 1982 ; Cesura et al., 1983 ; Davis et al., 1985 ; Mellerup et al., 1985; Wennogle et al., 1985; Biessen et al., 1990; Launay et al., 1992). Recently, it has been cloned and sequenced from rat brain (Blakely et al., 1991), rat basophilic leukemia cells (Hoffman et al., 1991), human brain (Lesch et al., 1993a), human platelets (Lesch et al., 1993b) and a human placental cell line (Ramamoorthy et al., 1993). Nevertheless, to our knowledge, no attempts have been made to characterize biochemically the 5-HT transporter from human brain and little information is available about the stoichiometry of its native form. Furthermore, the debated matter of site heterogeneity for the TCA [3H]-imipramine, the most used radio- ligand for labeling the 5-HT transporter, and, in general, the molecular relationships between TCAs, 299