The dopamine transporter (DAT) is a 12 transmembrane domain protein that mediates the re-uptake of the neurotransmitter dopamine (DA) into nerve terminals. Although DAT undergoes regulated trafficking in heterologous model systems, the mechanisms of trafficking to and retention at synapses are unknown. To determine regulators of DAT trafficking, we have adopted the C. elegans model system to utilize its powerful genetics and the ability for direct visualization of DA neurons in vivo and DAT-1 proteins in situ . To determine native DAT-1 distribution in situ , we raised antibodies against the DAT-1 carboxy terminus. To evaluate localization and trafficking of DAT-1 in vivo , we created a worm line (BY312) expressing a GFP:DAT-1 fusion protein driven by the
dat-1 promoter. To define synaptic localization, we created transgenic animals expressing mRFP fused to the C-terminus of the vesicular monoamine transporter (mRFP:VMAT). Localization of synaptic vesicles containing (VMAT) is dependent upon the activity of a kinesin motor protein (Unc-104). To examine the dependence of DAT-1 localization on Unc-104, we crossed BY312 animals into an UNC-104 deficient line (
unc-104(
e1265) ). Results: Immunofluorescence studies reveal staining of DAT-1 in wildtype (N2) animals with an enrichment of DAT-1 expression in synaptic regions that is lost in DAT-1 knockout animals (
dat-1 ). GFP:DAT-1 rescues a
dat-1 phenotype in vivo , and displays a distribution that is similar to that observed with antibody staining of N2 animals. Interestingly, DAT-1 localization appears unaffected in an
unc-104 knockout background. These findings suggest that DAT-1 accumulates at synaptic regions by a yet unknown mechanism. This localization is UNC-104 independent and distinct from pathways supporting VMAT localization. Experiments are underway to examine the mobility of GFP:DAT-1 in various cellular locations using fluorescent recovery after photobleaching (FRAP) and other fluorescent techniques. Individual NRSA (F31 NS46237-01) to PM and PPG (P01-DK58212) to RDB.