Inactivation of the human placental serotonin transporter by tyrosyl group-specific reagents.
Treatment of human placental brush-border membrane vesicles with tyrosyl group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole and tetranitromethane, inhibited NaCl gradient-driven serotonin uptake in these vesicles without affecting vesicle integrity. The concentrations of these reagents causing 50% inhibition of serotonin uptake were 3.75 mM, 10 microM and 5 microM, respectively. The inhibition of N-acetylimidazole was reversible with hydroxylamine and the inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was reversible with 2-mercaptoethanol. Kinetic analysis of serotonin uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the treatment decreased the maximal velocity of the transport system with virtually no effect on the affinity of the transporter for serotonin. Similarly, the treatment did not change the affinity of the transporter for Na+. Even though serotonin uptake was reduced in treated vesicles compared with control vesicles at all concentrations of Na+, in both cases the dependence of the uptake rate on Na+ concentration was hyperbolic, indicating the involvement of one Na+ per transport of one serotonin molecule. The serotonin transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. It is concluded that tyrosyl residues are essential for optimal transport function of the human placental serotonin transporter and that these critical tyrosyl residues are located at or near the Na+-binding site.
Ganapathy
V
V
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.
Kulanthaivel
P
P
Tiruppathi
C
C
Mahesh
V B
VB
Leibach
F H
FH
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008871:Microvilli / Q000187:drug effects* / Q000378:metabolism; D010920:Placenta / Q000187:drug effects / Q000378:metabolism; D011247:Pregnancy; D012701:Serotonin / Q000378:metabolism*; D012964:Sodium / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D007093:Imidazoles; D013439:Sulfhydryl Reagents; D012701:Serotonin; D014443:Tyrosine; D012964:Sodium; D009327:4-Chloro-7-nitrobenzofurazan; D013774:Tetranitromethane; C012160:N-acetylimidazole