The serotonin transporter-imipramine "receptor"

Abstract

The platelet plasma membrane serotonin transporter requires Na+ for two reactions, serotonin transport and imipramine binding. Although imipramine binding has been thought to reflect the same process required for serotonin binding prior to transport (Talvenheimo, J., Nelson, P.J., and Rudnick, G. (1979) J. Biol. Chem. 254, 4631-4635), binding and transport display markedly different responses to Na+. Imipramine binding (and competitive inhibition of transport) apparently requires two sodium ions which bind with a KD of 300 +/- 70 meq/liter. The total number of sites (Bmax) is the same at all Na+ concentrations, but the affinity for imipramine increases from 7.3 x 10(6) M-1 at 20 meq/liter to 110 x 10(6) M-1 at 200 meq/liter. Na+ acts, at least in part, by decreasing the rate of imipramine dissociation from its binding site. Serotonin binding displaces imipramine from its site on the membrane. In contrast to imipramine binding, this displacement is a simple, hyperbolic function of Na+ concentration with a KD for Na+ of 400 +/- 100 meq/liter, which suggests that only one Na+ is required. Serotonin transport is also much less responsive to Na+ concentration. Over the same concentration range in which the affinity for imipramine increases 15-fold, the affinity for serotonin increases only 2-fold. Despite the lack of Na+ effect on the Bmax for imipramine binding, the Vmax for serotonin transport increases as a simple saturable function of Na+ with a KM (Na+) of 52 meq/liter. Thus, substrate translocation as well as binding requires Na+. Since serotonin is cotransported with Na+, the serotonin gradient accumulated depends on the coupling stoichiometry and the magnitude of the Na+ gradient imposed. From the response of the serotonin gradient to imposed Na+ gradients, we calculated a serotonin:Na+ cotransport stoichiometry of 0.9. Taken together, the results suggest that serotonin and imipramine bind either to the same site or to mutually exclusive sites, but maximal imipramine binding requires two sodium ions, while maximal serotonin binding and translocation requires only one.