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.
Highlights
The platelet plasma membrane serotonin transpohrtoerrmones (Briley et a t, 1979)
Imipramine bind- anism of neurotransmitterre-uptake(Talvenheimo et al, ing apparently requires two sodium ions which bind with a KO of 300 f 70 meq/liter
At each Na’ concentration, imipramine binding saturates with simple hyperbolic behavior suggesting a single class of sites
Summary
6115-6119, I983 Printed in U.S.A. DIFFERENT SODIUM REQUIREMENTS FOR IMIPRAMINE BINDING AND SEROTONIN TRANSLOCATION*. Imipramine bind- anism of neurotransmitterre-uptake(Talvenheimo et al, ing (and competitive inhibition of transport) apparently requires two sodium ions which bind with a KO of 300 f 70 meq/liter. Despite mal binding of imipramine requires two sodium ions, thelack of Na+effectonthe B,, forimipramine binding, the V,,, for serotonin transport increases as a simple saturable functionof Na+ with a K M(Na+)of. Imipramine binding was first measured in platelet plasma membranes (Rudnick and Talvenheimo, 1978; Talvenheimo et al, 1979) andsubsequentlyatsitesinbrainandintact ously (Talvenheimo et al, 1979). Imipramine bindinwg as assayed platelets (Raisman et al, 1979; Paul et al, 1980) which have in 100 mM sodium phosphate, pH 6.7, containing 100 mM NaC1, 1 been nominated as receptors for endogenous “mood control” mM MgSO,, andthe indicated concentration of [3H]imipramine.
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