This application requests renewal of an Independent Scientist Award (ISA) to support Prof James O. Schenk in the Depts of Chemistry and Biochemistry/Biophysics at Washington State University (WSU). The ISA will enhance Prof Schenk's career and professional growth because it will continue to: (a) reduce his administrative and teaching responsibilities, (b) enhance development of experimental approaches to problems in drug abuse, (C) allow maximal efforts using the new models developed during the previously funded period, and (d) provide opportunities for expanding the scope of his research through enhanced and continuing collaborations with others in the field. During the previously funded period Prof Schenk developed a rotating disk voltammetric method to measure time-resolved dopamine (DA) release and reuptake in tissue preparations of striatum, nucleus accumbens, and medial prefrontal cortex as well as in a cell culture system expressing the human dopamine transporter (DAT). The results obtained include proposing multisubstrate mechanisms of DA transport in A9 and A10 DA terminal areas, new insights into the chemistry of DA transport via studies of structure-activity and D2O-solvent isotope effects, an approach to measuring the pre-steady state binding of DA to the actively transporting DAT, evidence that DAT activity may be controlled by presynaptic autoreceptors, development of a kinetic model to distinguish binding of structurally similar and dissimilar DA transport inhibitors, and mechanistic insights on changes in the function of DAT following withdrawal from contingent and non-contingent treatments with cocaine. In addition, the technique has been shared with other interested research groups including the laboratories of Prof B. Sorg (WSU), Prof P. Kalivas (WSU), Prof. J. Justice (Emory University), Prof. Diane Latteman (University of WA, Seattle), Prof. R. N. Adams (Univ. of KS) and the labs of Profs. K. Neve, A. Janowsky, and A. Eshleman at the VA Medical Center at the Oregon Health Sciences Univ. (Portland, OR). Work proposed for the next period of research includes, correlations of DA transport at DAT with intracellular changes in ion cosubstrate (Na+ and Cl-) concentrations, continued in vivo and in vitro studies of DA transport mechanisms and actions of inhibitors in DA terminal regions of the rat, studies of human DAT mechanisms and actions of inhibitors in expression systems (such as in HEK-293 cells and N2A cells), studies of bi-directional transport of substrates by DAT, and studies of the action of second messengers on DAT functioning.
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