Maternal-facing brush border membranes prepared from normal term human placentas possess the norepinephrine transporter. We investigated the interaction of nisoxetine with the human norepinephrine transporter by examining the binding of this ligand to the placental brush border membranes. Scatchard analysis revealed that nisoxetine bound with high affinity to a single class of binding sites in the membranes (dissociation constant = 13.8 ± 0.4 nM). This value obtained from equilibrium experiments matched the value (11.2 nM) which was calculated using the association and dissociation rate constants. The maximal binding capacity (Bmax) was 5.1 ± 0.1 pmol/mg of protein. The binding exhibited an absolute requirement for Na+ as well as Ch. Presence of these ions enhanced the binding affinity without affecting Bmax. Kinetic analyses revealed that the coupling ratio of Na+/nisoxetine was 2, whereas the coupling ratio of Cl−/nisoxetine was 1. The binding was most potently inhibited by the ligands of the norepinephrine transporter (desipramine and nomifensine). The ligands of the serotonin transporter (imipramine, paroxetine, and fluoxetine) showed intermediate inhibitory potencies, whereas the ligands of the dopamine transporter (bupropion and GBR 12909) were the least potent. Among the monoamines, dopamine was the most potent inhibitor, followed by norepinephrine and serotonin. Though both cocaine and its analog RTI-55 were powerful inhibitors of the binding, RTI-55 was ≈150 times more effective than cocaine. The inhibition of binding by norepinephrine, cocaine, and RTI-55 was competitive. Uptake of norepinephrine measured in membrane vesicles was found to be inhibited by treatment of the vesicles with phenylarsine oxide, a reagent specific for vicinal dithiol groups. The transporter was protected to a significant extent from this inhibition if norepinephrine or nisoxetine was present during treatment with the reagent, indicating that the binding sites on the transporter for norepinephrine and nisoxetine are either identical or considerably overlap with each other. The binding of nisoxetine to the membranes was pH dependent with a pKa of 5.7. Analysis of the influence of pH on the kinetic parameters for nisoxetine binding with respect to the dependence of binding on the concentration of Na+, Cl−, and nisoxetine showed that H+ decreased the affinity of the transporter for Na+, Cl−, and nisoxetine, but did not influence Bmax, nor did it alter the Na+/nisoxetine and Cl−/nisoxetine coupling ratios. K+ showed a small, but significant, stimulatory effect on the binding. Zn2+ at concentrations ≤100 µM significantly stimulated the binding but caused inhibition at higher concentrations. This detailed report on the characteristics of the binding of nisoxetine to the norepinephrine transpoprter from a tissue of human origin show that nisoxetine can be used as a specifc, high-affinity ligand in studies involving the human norepinephrine transporter.
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