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 [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.
Rotondo
A
A
Institute of Psychiatry, University of Pisa, Italy.
Giannaccini
G
G
Betti
L
L
Chiellini
G
G
Marazziti
D
D
Martin
C
C
Lucacchini
A
A
Cassano
G B
GB
D000929:Antidepressive Agents, Tricyclic; D001923:Brain Chemistry / Q000502:physiology*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007099:Imipramine; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008566:Membranes / Q000378:metabolism; D008875:Middle Aged; D017072:Neostriatum / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017374:Paroxetine; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors
D000929:Antidepressive Agents, Tricyclic; D002352:Carrier Proteins; D007099:Imipramine; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D017374:Paroxetine; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human