Substrate and inhibitor binding and translocation by the platelet plasma membrane serotonin transporter.
The Na+ and Cl- dependence of imipramine binding and dissociation were determined in platelet plasma membrane vesicles. Equilibrium imipramine binding affinity depends on Na+ binding to two non-interacting, low-affinity sites. Binding of a single Cl- ion also enhances imipramine affinity. Imipramine dissociation is inhibited by Na+ and Cl-, indicating that both ions can bind after imipramine. Of the two Na+ ions required for imipramine binding, only one is involved in slowing imipramine dissociation, indicating that imipramine binding makes the two Na+ ions non-equivalent. The initial rate of imipramine association is strongly Na(+)-dependent, suggesting that Na+ binds prior to imipramine. Cl-, however, affects imipramine dissociation but not association. Thus, while Na+ and Cl- can bind either before or after imipramine, kinetic considerations impose a most likely binding order of first Na+, then imipramine and finally Cl-. We have confirmed and extended these conclusions using serotonin exchange and efflux measurements. Efflux of radioactivity from vesicles preloaded with [3H]serotonin is stimulated by both external K+ and external unlabelled serotonin. K+ acts to accelerate a step that is rate-limiting for net efflux but that does not involve Na+, Cl- or serotonin translocation. Unlabelled serotonin accelerates radioactivity efflux by exchanging with intravesicular label. This serotonin exchange requires external Cl-, but not external Na+. These results suggest that first Na+, then serotonin and finally Cl- bind from the external medium. Although serotonin exchange requires external Cl-, internal Cl- is not required. These results suggest that translocation does not disturb the spatial order of bound substrates, which dissociate internally in a first-in-first-out order.
Humphreys
C J
CJ
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510-8066.
Beidler
D
D
Rudnick
G
G
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007099:Imipramine / Q000097:blood; D007700:Kinetics; D012701:Serotonin / Q000097:blood*; D050486:Serotonin Plasma Membrane Transport Proteins
D001798:Blood Proteins; D002352:Carrier Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D007099:Imipramine