PMID- 27638414 OWN - NLM STAT- MEDLINE DCOM- 20170509 LR - 20180406 IS - 1873-2968 (Electronic) IS - 0006-2952 (Linking) VI - 120 DP - 2016 Nov 15 TI - The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux. PG - 46-55 LID - S0006-2952(16)30268-4 [pii] LID - 10.1016/j.bcp.2016.09.006 [doi] AB - The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser. CI - Copyright (c) 2016 Elsevier Inc. All rights reserved. FAU - Sealover, Natalie R AU - Sealover NR AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, IN 47907, United States. FAU - Felts, Bruce AU - Felts B AD - Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States. FAU - Kuntz, Charles P AU - Kuntz CP AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, IN 47907, United States. FAU - Jarrard, Rachel E AU - Jarrard RE AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, IN 47907, United States. FAU - Hockerman, Gregory H AU - Hockerman GH AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, IN 47907, United States. FAU - Lamb, Patrick W AU - Lamb PW FAU - Barker, Eric L AU - Barker EL AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, IN 47907, United States. Electronic address: barkerel@purdue.edu. FAU - Henry, L Keith AU - Henry LK AD - Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States. Electronic address: keith.henry@med.und.edu. LA - eng GR - R01 DA018682/DA/NIDA NIH HHS/United States PT - Comparative Study PT - Journal Article DEP - 20160913 PL - England TA - Biochem Pharmacol JT - Biochemical pharmacology JID - 0101032 RN - 0 (Caenorhabditis elegans Proteins) RN - 0 (Drosophila Proteins) RN - 0 (Hallucinogens) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Fusion Proteins) RN - 0 (SLC6A4 protein, human) RN - 0 (SerT protein, Drosophila) RN - 0 (Serotonin Agents) RN - 0 (Serotonin Plasma Membrane Transport Proteins) RN - 0 (mod-5 protein, C elegans) RN - 333DO1RDJY (Serotonin) RN - KE1SEN21RM (N-Methyl-3,4-methylenedioxyamphetamine) SB - IM EIN - Biochem Pharmacol. 2017 Nov 28;147:9. PMID: 29195124 MH - Amino Acid Substitution MH - Animals MH - Caenorhabditis elegans Proteins/chemistry/genetics/*metabolism MH - Drosophila Proteins/chemistry/genetics/*metabolism MH - Drosophila melanogaster MH - HEK293 Cells MH - Hallucinogens/*metabolism/pharmacology MH - Humans MH - Mutagenesis, Site-Directed MH - Mutation MH - N-Methyl-3,4-methylenedioxyamphetamine/*metabolism/pharmacology MH - Oocytes/drug effects/metabolism MH - Patch-Clamp Techniques MH - Peptide Fragments/chemistry/genetics/metabolism MH - Protein Interaction Domains and Motifs MH - Recombinant Fusion Proteins/chemistry/metabolism MH - Serotonin/metabolism MH - Serotonin Agents/*metabolism/pharmacology MH - Serotonin Plasma Membrane Transport Proteins/chemistry/genetics/*metabolism MH - Species Specificity MH - Substrate Specificity MH - Xenopus laevis OTO - NOTNLM OT - Amphetamine OT - Efflux OT - Electrophysiology OT - Mutagenesis OT - Serotonin transporter EDAT- 2016/10/26 06:00 MHDA- 2017/05/10 06:00 CRDT- 2016/09/18 06:00 PHST- 2016/07/15 00:00 [received] PHST- 2016/09/08 00:00 [accepted] PHST- 2016/10/26 06:00 [pubmed] PHST- 2017/05/10 06:00 [medline] PHST- 2016/09/18 06:00 [entrez] AID - S0006-2952(16)30268-4 [pii] AID - 10.1016/j.bcp.2016.09.006 [doi] PST - ppublish SO - Biochem Pharmacol. 2016 Nov 15;120:46-55. doi: 10.1016/j.bcp.2016.09.006. Epub 2016 Sep 13.