A genetic screen in Caenorhabditis elegans for dopamine neuron insensitivity to 6-hydroxydopamine identifies dopamine transporter mutants impacting transporter biosynthesis and trafficking.
The presynaptic dopamine (DA) transporter (DAT) is a major determinant of synaptic DA inactivation, an important target for psychostimulants including cocaine and amphetamine, and a mediator of DA neuron vulnerability to the neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion. To exploit genetic approaches for the study of DATs and neural degeneration, we exploited the visibility of green fluorescent protein (GFP)-tagged DA neurons in transgenic nematodes to implement a forward genetic screen for suppressors of 6-OHDA sensitivity. In our initial effort, we identified three novel dat-1 alleles conferring 6-OHDA resistance. Two of the dat-1 alleles derive from point mutations in conserved glycine residues (G55, G90) in contiguous DAT-1 transmembrane domains (TM1 and TM2, respectively), whereas the third allele results in altered translation of the transporter's COOH terminus. Our studies reveal biosynthetic, trafficking and functional defects in the DAT-1 mutants, exhibited both in vitro and in vivo. These studies validate a forward genetic approach to the isolation of DA neuron-specific toxin suppressors and point to critical contributions of the mutated residues, as well as elements of the DAT-1 COOH terminus, to functional expression of catecholamine transporters in neurons.
Department of Anesthesiology, Vanderbilt School of Medicine, Nashville, Tennessee 37232-8548, USA.
Richard
R
Nass
Maureen K
MK
Hahn
Tammy
T
Jessen
Paul W
PW
McDonald
Lucia
L
Carvelli
Randy D
RD
Blakely
D018663:Adrenergic Agents / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western / Q000379:methods; D017173:Caenorhabditis elegans; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D005820:Genetic Testing / Q000379:methods; D023281:Genomics / Q000379:methods; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D008958:Models, Molecular; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D009474:Neurons / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D016627:Oxidopamine / Q000494:pharmacology; D021381:Protein Transport / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism
D018663:Adrenergic Agents; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014316:Tritium; D049452:Green Fluorescent Proteins; D016627:Oxidopamine; D004298:Dopamine