MDMA ("ecstasy") has been widely reported as a drug of abuse and as a neurotoxin. This report describes the mechanism ofMDMA action at serotonin transporters from plasma membranes and secretory vesicles. MDMA stimulates serotonin efflux from both types of membrane vesicle. In plasma membrane vesicles isolated from human platelets, MDMA inhibits serotonin transport and [3H]imipramine binding by direct interaction with the Na+-dependent serotonin transporter. MDMA stimulates radiolabel efflux from plasma membrane vesicles preloaded with PHlserotonin in a stereospecific, Na+-dependent, and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffmi granules, which contain the vesicular biogenic amine transporter, MDMA inhibits ATP-dependent [3H]serotonin accumulation and stimulates efflux of previously accumulated [3H]serotonin.Stimulation of vesicular [3Hlserotonin efflux is due to dissipation of the transmembrane pH difference generated by ATP hydrolysis and to direct interaction with the vesicular amine transporter.Serotonin transport has been implicated in the mechanism of a number of amphetamine derivatives including p-chloroamphetamine, fenfluramine, 3,4-methylenedioxyamphetamine, and MDMA. These compounds cause an acute release of serotonin in vivo (1, 2) and in vitro (3-5) and also lead to a long-term depletion of serotonin (6, 7) that correlates with morphological damage to serotonergic nerve endings (7-10). The serotonin transporter has been implicated in these phenomena since inhibitors of serotonin transport block the effect of amphetamine derivatives on acute serotonin release and destruction of serotonergic terminals (5,11,12). These results suggest that the serotonin transporter either mediates the entry of neurotoxic amphetamines into serotonergic terminals or participates in sequelae leading to serotonin release and depletion (13) or both.The observation that Ca2+ is not required for amphetamine-induced serotonin release (4) suggests that exocytosis is not involved. Thus, it is likely that amphetamines may induce release by reversal of the transport systems that normally catalyze accumulation of serotonin to high levels within the neuron and the synaptic vesicle. We, therefore, examined two membrane vesicle model systems for serotonin transport to determine if MDMA directly affects these transport systems. Serotonergic neurons, like other cells that secrete serotonin, contain two serotonin transport systems that function in series (14). One of these systems transports serotonin into the cell, and the other sequesters intracellular serotonin within secretory vesicles.Purified platelet plasma membrane vesicles contain the Na+-dependent imipramine-sensitive serotonin transporter responsible for serotonin reuptake into presynaptic nerve endings (15). When appropriate transmembrane ion gradients are imposed, these vesicles accumulate [3H]serotonin to concentrations several hundredfold higher than in the external medium...

We prepared membrane vesicles from stable LLC-PK 1 cells expressing serotonin (5-HT) ␥-aminobutyric acid (GABA) and norepinephrine (NE) transporters (SERT, GAT-1, and NET).

The effects of Na+ and Cl- on the binding of [3H]imipramine and the cocaine analog [125I]-beta-carbomethoxy-3 beta-(4-iodophenyl)tropane([125I]-beta-CIT) to the human platelet serotonin transporter have been measured. The ion dependence of beta-CIT binding is consistent with binding beta-CIT together with one Na+ ion, but not in an ordered sequence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This conclusion is based on the observation that both imipramine association rate constants and equilibrium affinity constants show a sigmoidal Na+ dependence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently by slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nontransported inhibitors, such as imipramine and citalopram, as well as the transport substrates serotonin and 3,4-(methylenedioxy)methamphetamine all displaced beta-CIT binding in the absence of NaCl. In the absence of Cl-, Na+ increased the affinity of nontransported inhibitors but not of substrates. The results suggest that Na+ and Cl- induce independent changes in the transporter binding site and that binding of substrates and inhibitors is affected differently by these changes.

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p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.