The dopamine (DA) transporter (DAT) is a polytrophic, membrane protein that utilizes the co-transport of Na+ and Cl- ions to energize the rapid re-uptake of DA from the synapse in order to terminate extracellular DA signalling and facilitate re-release. As worm synaptic structure in general, and the expression of DAT in particular, are conserved across phyla, studies with the orthologous Caenorhabditis elegans protein model provide an ideal opportunity to probe fundamental questions of transporter structure and function in an in vivo setting using powerful forward and reverse genetic screens. The Million Mutation Project (MMP, http://genome.sfu.ca/mmp/) represents a library of ~2,000 mutagenized worm strains where sequencing at a depth of 15X genome coverage reveals the presence of, on average, ~9 new non-synonymous alleles per gene,whose characterization can reveal novel links to protein structure and function. Thirteen such coding variants are present in the MMP. We have initiated the functional characterization of these variants, focusing initially on five lines that bear amino acid changes (D8N, P9L, M20T, P596S, P609S) at highly conserved locations and/or that are associated with regions suspected to confer/support DAT activity or regulation based on mammalian DAT studies. To date, we have confirmed the behavior of four of these lines consistent with DAT-1 loss of function, assessed by the presence of Swimming-induced paralysis (Swip). Results from the successful genetic cross of mutant worms with cat-2 tyrosine hydroxylase (TH, cat-2) loss of function mutant lines and the introduction of MMP mutations into a GFP-DAT-1 expression construct via site-directed mutagenesis of the wildtype dat-1 will be used to determine the impact of dat-1 missense alleles on in vivo DAT function and distribution in DA neurons. Supported by NIH award MH095055 (RDB) and NSF award 1505176 (PF).