The Antidepressant-Sensitive Dopamine Transporter inDrosophila melanogaster: A Primordial Carrier for Catecholamines
- 1Vollum Institute (P.P., M.S.S., S.G.A.) and 2Howard Hughes Medical Institute (S.G.A.), Oregon Health Sciences University, Portland, Oregon; and 3Department of Biology, University of Virginia, Charlottesville, Virginia (S.K.P., J.H.)
Abstract
Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 μM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian catecholamine transporters. The combination of a DAT-like substrate selectivity and norepinephrine transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral catecholamine transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior.
Footnotes
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Send reprint requests to: Peter Pörzgen or Susan G. Amara, 3181 SW Sam Jackson Park Rd., OHSU, Vollum Institute L-474, Portland, OR, 97201. Email: poerzgen{at}ohsu.edu;amaras{at}ohsu.edu
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This work was supported by Grants from NARSAD (M.S.S.), the National Institute on Drug Abuse (DA07595 and DA12408) (S.G.A.), National Institutes of Health Grant GM/DA27318 (J.H.), and the Deutsche Forschungsgemeinschaft (P.P.).
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Parts of this work were presented at the 29th Annual Meeting of the Society for Neuroscience (Soc Neurosci1999;25:160).
- Abbreviations:
- DA
- dopamine
- NE
- (−)-norepinephrine
- 5-HT
- 5-hydroxytryptamine (serotonin)
- DAT
- dopamine transporter
- SERT
- serotonin transporter
- NET
- norepinephrine transporter
- TA
- p-tyramine
- dSERT
- Drosophila melanogaster serotonin transporter
- OA
- octopamine
- TMD
- transmembrane domain
- ceDAT
- Caenorhabditiselegans dopamine transporter
- dDAT
- Drosophila melanogaster dopamine transporter
- NTT
- neurotransmitter transporter
- hDAT
- human dopamine transporter
- bp
- base pair(s)
- kb
- kilobase pair(s)
- EST
- expressed sequence tag
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- COS-7
- SV40-transformed African green monkey kidney cells
- MDCK
- Madin-Darby canine kidney
- KRH
- Krebs-Ringer's-HEPES
- hSERT
- human serotonin transporter
- hNET
- human norepinephrine transporter
- EL
- extracellular loop
- PK-C
- protein kinase C
- AA
- amino acid
- CNS
- central nervous system
- Epi
- (−)-epinephrine
- fET
- frog epinephrine transporter
- TCA
- tricyclic antidepressant
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- Received August 16, 2000.
- Accepted October 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
