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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.