Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by 3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected 3H]5-HT uptake and 3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished 3H]5-HT uptake only minimally reduced 3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet 3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on 3H]IMI binding and 3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of 3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential 3H]5-HT uptake blockers. 5-HT was a weak displacer of 3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the 3H]IMI binding site and the 5-HT transporter complex in human platelets.