Cocaine initiates its euphoric effects in the brain by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. No specific DAT-ligand contacts have been identified to date, but it is widely held (owing to a previous report) that the DAT transmembrane (TM) 1 aspartic acid residue (D79) forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine that governs recognition of the ligand. Alternatively, the D79 residue may contribute to a ligand aromatic binding pocket, an ion permeation pore that influences ligand binding, or may simply form intramolecular contacts that retain DAT infrastructure. Toward the goal of elaborating on the nature of DAT substrate and inhibtor binding sites, it is critical to determine whether the "ion pair" model for DAT-ligand interactions is credible. The objective of this proposal is to address the validity of the ion pair model, while also assessing alternative potential contributions of the D79 residue to DAT structure and function. D79 DAT mutants that modify side chain size, charge or hydrogen bonding potential will be pharmacologically characterized with a diverse collection of DAT substrates and inhibitors. Other, cysteine-substituted, DAT mutants will test the accessibility of a given position in the DAT polypeptide to cysteine-specific alkylating agents, and whether such alkylation events influence DAT function. By measuring accessibility of an introduced DAT TM 1 (or vicinity) cysteine residue (variably located) in the presence and absence of substrates or inhibitors, the spatial relationship between D79 and DAT substrates and inhibitors will be explored. Because the TM 1 aspartate has been implicated as a contributor to the substrate permeation pore, the substituted cysteine accessibility method will also be used to elaborate on the secondary structure and cell membrane orientation of TM 1 in the vicinity of D79, for which there are multiple models. The proposed experiments should significantly enhance understanding of the TM 1 aspartate residue role in DAT function, as well as advance structure-function studies on the plasma membrane norepinephrine and serotonin transporters which share this residue. Clarification of the role of this residue in recognition of dopamine and cocaine may forward rational design of therapeutics that block cocaine action without significantly interfering with dopamine uptake. [unreadable] [unreadable]