Polar amino acids lying within three hydrophobic regions of the dopamine transporter (DAT) are analogous to those important for ligand recognition by catechoamilne receptors. Possible functional significance of these amino adds was examined by expressing DAT cDNAs mutated in these polar residues. Replacement of aspartate at position 79 with alanine, glycine, or glutamate dramatically reduced uptake of binding. These results demonstrate that aspartate and serine residues lying within the first and seventh hydrophobic putative transmembrane regions are crucial for DAT function and provide identification of residues differentially important for cocaine binding and for dopamine uptake.The dopamine transporter (DAT) aids in terminating dopaminergic neurotransmission by sodium-dependent reaccumulation of released dopamine into presynaptic neurons (1, 2) and is key for actions of cocaine and dopamine-specific neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) (3,4). cDNA cloning studies have recently elucidated the primary structures of DATs and other homologous members of the sodiumdependent neurotransmitter (plus) transporter family that are expressed in brain or kidney (5-12). However, the transporter sites recognizing neurotransmitters, sodium, neurotoxins, and drugs, including cocaine, are unknown. Clues as to DAT regions that could participate in dopamine recognition could come from studies of catecholamine binding to mutant adrenergic receptors (13, 14), which implicate aspartic acid and serine residues lying in hydrophobic regions in catecholamine binding. DAT contains a single aspartic acid residue in its first hydrophobic domain and serine residues in its seventh and eighth hydrophobic domains. We now report selective effects of mutations of these residues on DAT dopamine uptake and cocaine analog recognition. To more directly compare influences on these two end points, we have examined both binding and uptake in intact cell preparations. MATERIALS AND METHODS Single-stranded template for mutagenesis was derived from pcDNADAT1 (5); confirmed by DNA sequencing; and mutated by annealing oligonucleotides corresponding to the mutant sequences (Fig. 1)

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