A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine.
Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.
Greer
Christina L
CL
Department of Psychiatry and Biobehavioral Sciences, The David Geffen School of Medicine at UCLA, Gonda (Goldschmied) Center for Genetic and Neuroscience Research, 695 Charles Young Drive, Los Angeles, California 90095-1761, USA.
Grygoruk
Anna
A
Patton
David E
DE
Ley
Brett
B
Romero-Calderon
Rafael
R
Chang
Hui-Yun
HY
Houshyar
Roozbeh
R
Bainton
Roland J
RJ
Diantonio
Aaron
A
Krantz
David E
DE
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002522:Chlorocebus aethiops; D004298:Dopamine / Q000378:metabolism*; D004330:Drosophila; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000502:physiology; D009655:Octopamine / Q000378:metabolism*; D011078:Polychlorinated Biphenyls / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D012110:Reserpine / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism*; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D018759:Adrenergic Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D020033:Protein Isoforms; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D009655:Octopamine; D012701:Serotonin; D012110:Reserpine; D011078:Polychlorinated Biphenyls; D004298:Dopamine