The transporters for dopamine, norepinephrine, and serotonin are targets for drugs of abuse such as cocaine and amphetamine, and for therapeutic drugs such as the antidepressants. The structure and mechanism of these membrane spanning protein remains unknown. However, the recent cloning and expression of these transporters has allowed progress to be made in better understanding how these proteins work. The proposed experiments build on this recent progress by combining the developments in molecular biology with recent advances in electrochemical measurement technology. The kinetics and mechanism of the human norepinephrine transporter will be examined with rapid voltammetric methods. Hypotheses concerning complimentary alterations in substrate structure and mutation in the transporter will be tested. Interactions of the amino group of the substrate with an amino acid residue in transmembrane region one constitute one set of experiments. Interactions of the ring hydroxyls with residues in transmembrane region seven compromise a second set of experiments. An additional experiment is designed to characterize the transport characteristics of a substrate designed to act as photoaffinity label for probing the active site of transport.
