One-step purification of the serotonin transporter located at the human platelet plasma membrane.
A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.
Launay
J M
JM
Claude Bernard Neurochimie des Communications Cellulaires, Hôpital Saint-Louis, Paris, France.
Geoffroy
C
C
Mutel
V
V
Buckle
M
M
Cesura
A
A
Alouf
J E
JE
Da Prada
M
M
D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010042:Ouabain / Q000378:metabolism; D017374:Paroxetine; D010880:Piperidines / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists; D050486:Serotonin Plasma Membrane Transport Proteins; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000378:metabolism
D001798:Blood Proteins; D002352:Carrier Proteins; D010880:Piperidines; D012702:Serotonin Antagonists; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; C032811:dihydrotetrabenazine; D017374:Paroxetine; D010042:Ouabain; D013747:Tetrabenazine