During the previous grant period the laboratory used the first cDNA encoding a cocaine-sensitive biogenic amine transporter, the human norepinephrine transporter (NET), as the basis for the isolation and characterization of a cDNA encoding the dopamine transporter (DAT) which has been proposed to be the most significant site of action for the euphoric and addictive effects of cocaine. Although many neurobiological issues need to be investigated before the biology of psychomotor stimulant addiction can be explained, the studies proposed in this continuing renewal application will continue to build on the view that significant insights will come from the fundamental biology of the transporters themselves. This proposal outlines plans for the further exploration of basic structure-function relationships, electrogenic properties and cell biological regulation of DAT and NET, areas that provided some of the most interesting observations during the previous grant period. Continuing studies using chimeras, selected point mutants and cysteine-scanning and modification (SCAM) approaches are being coupled with kinetic analyses of transport, electrophysiological approaches and binding studies to further probe the structure of substrate and antagonist binding sites, as well as the permeation pathway for ions. Recent work from this laboratory has shown that dopamine uptake by DAT is an electrogenic and voltage-dependent process. Furthermore, members of Na+/CI- -dependent family of neurotransmitter transporters, including DAT, can mediate macroscopic ionic currents which are not stoichiometrically linked to substrate movement. The mechanism and consequences of these currents, in particular their potential to activate voltage-gated calcium channels and intracellular signaling pathways, will be examined. The proposed experiments also aim to examine how intracellular signaling mechanisms regulate the number of carriers expressed at the cell surface, focusing on a particularly striking effect of PKC activation on the internalization and sequestration of NET and DAT proteins. Our overall goal is to relate protein structural features to novel functional properties and regulatory mechanisms, and to establish the contributions of the DA and NE carriers to neuronal function.
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