Dithiothreithol promotes a higher affinity state of the serotonin transporter for the tricyclic antidepressant, imipramine.

Neurotransmitter transporters are present in nerve terminal membranes and are responsible for the inactivation of released neurotransmitter by reuptake. One such transporter is the serotonin transporter which also represents the site of action of many clinically used tricyclic antidepressants, such as imipramine. Recently, most of the known Na+-dependent neurotransmitter transporters have been cloned and sequenced (for review see (I)). On the basis of sequence similarities, the transporters can be divided into two superfamilies. Within each family many features are conserved which may serve important structural, functional or regulatory roles. One such feature, conserved throughout the superfamily of which the serotonin transporter is a member, is two cysteines, nine amino acids apart, which are located on a putative large extracellular loop of the protein. It has been proposed that these cysteines may be involved in ligand binding and/or substrate uptake by the transporters, or perhaps function in maintaining the transporters in an active conformation (2). Thus, the effects of the sulphydryl reducing agent, dithiothreithol (DTT), on ligand binding to the human platelet serotonin transporter were studied. When the binding of the tricyclic antidepressant, [3H]imipramine, to human platelet membranes was measured over a range of increasing DTT concentrations, a concentration dependent increase in [3H]imipramine binding was found with maximum binding being achieved at a DTT concentration of 5 mM. Saturation studies were performed, incubating platelet membranes with a range of [3H]imipramine concentrations in the presence of DTT. Analysis of the saturation curves revealed a 2 to 3 fold decrease in the apparent KD value for [3H]imipramine binding to membranes in the presence of DTT, relative to the control value determined in the absence of DTT. This DTT induced decrease in the KD value was B,, values were unaffected by the presence of DTT (Table 1). The kinetic basis of this DTT induced increase in affinity of the platelet membranes for [3H]imipramine was also studied via dilution-induced dissociation experiments, as described previously (3). Human platelet membranes and [3H]imipramine were incubated in the presence of 10 mM DTT and diluted 50-fold into assay buffer containing 10 mM DTT. A slowing of the rate of dissociation of [3H]imipramine from the membranes was observed relative to the control experiment, which was carried out in the absence of DTT. The dissociation curve was biphasic in the absence of DTT while in the presence of DTT the curve was either monophasic (slowly dissociating) or tended towards this condition (Fig.1.). The dissociation rate of [3H]imipramine from platelet membranes was measured over a range of DTT concentrations. As the DTT concentrations increased, there was an apparent concentrationdependent shift in the [3H]imipramine dissociation curve from the