Presynaptic dopamine (DA) transporters (DATs) constitute the primary mechanism for inactivation of DA in the brain. DAT proteins are high- affinity targets for important addictive and therapeutic drugs including cocaine, amphetamines and methylphenidate (Ritalin/TM). Little is known regarding how DAT proteins form a selective permeation pathway for DA and how different antagonist impact activity. Recent studies reveal DATs to be acutely regulated by coordinated mechanisms involving kinase activation, transporter phosphorylation and altered membrane trafficking/stabilization, though as yet genes responsible for this regulation remain to be identified. In rodents and man, DATs also constitute the portal through which exogenous neurotoxins (e.g. 6- OHDA, methamphetamine and MPP+) enter these neurons and affect lesions reminiscent of the selective pathology of Parkinson's disease. Controversy exits as to whether these toxic insults trigger cell death in vivo via necrotic or apoptotic pathways. We have cloned the product of the C. elegans gene T23G5.5 and demonstrated in its function as a DA transporter (CeDAT), opening the door to genetic strategies for the definition of critical DAT residues, the identification of CeDAT regulators and an understanding of molecular contributors to dopaminergic neuron sensitivity to environmental toxins. In our proposal, we seek to 1) validate the molecular and cellular specificity of CeDAT- targeted drugs and toxins, evaluate the properties of 6-OHDA-induced DA neuron degeneration in the work and determine whether CeDAT is both necessary and sufficient for toxin sensitivity, 2) to establish the cellular specificity, developmental expression and subcellular localization of CeDAT protein CeDAT mutants and CeDAT regulators using novel gain-of-function screens involving suppression of sensitivity to 6-OHDA. Together, these efforts provide important opportunities to identify and characterize regulators of transporter expression, localization and function and may provide clues to molecular determinants of dopamine- dependent psychiatric and neurodegenerative syndromes.