In vivo analysis of a salt bridge at the external gate of the Drosophila melanogaster serotonin transporter in response to amphetamines

Monoamine neurotransmitter transporters are membrane proteins responsible for the clearing of biogenic amines from a synapse. These transporters are targets for many important pharmaceuticals including antidepressants, as well as psychostimulant drugs such as cocaine and amphetamines. Amphetamines are believed to elicit their psychostimulant activity primarily by inducing a reversal of the transport cycle and increasing neurotransmitter release into the synapse, though the mechanism of this activity is incompletely understood. Previous in vitro research has suggested functional significance of a conserved salt bridge in the serotonin transporter (SERT) in amphetamine-induced 5-HT efflux. This salt bridge is disrupted in the Drosophila melanogaster SERT. Here, a mutant line of D. melanogaster expressing a SERT with a restored salt bridge (dSERT N484D) was studied. Changes in neurochemistry induced by methamphetamine (METH) or 3, 4-methylenedioxymethamphetamine (MDMA) were examined in vivo. HPLC/Mass spectrometry was used to quantify brain concentrations of neurotransmitters in fly tissue after drug treatment. N484D flies were found to have significantly depleted 5-HT in response to 0.05% MDMA relative to wild-type. This depletion of 5-HT was not observed after treatment with 0.6% MDMA or 0.6% METH. No significant drug-induced changes were observed in concentrations of other neurotransmitters examined. The results show that the presence of the salt bridge at the external gate of SERT may be important for amphetamine-induced 5-HT efflux, and helps explain pharmacological differences observed between hSERT and dSERT