The strategy of this research proposal is based on the rationale that a basic understanding of the mechanism of action of cocaine both centrally and peripherally depends upon the biochemical characterization of the proteins which bind this drug. This proposal involves the development and use of novel radioiodinated photoaffinity probes derived from cocaine, from 2beta-carbomethoxy-3beta- (4-fluorophenyl)tropane (CFT), and from specific monoamine uptake inhibitors (i.e., imipramine, desimipramine). These probes will be used to photolabel monoamine transporters in rat striatal synaptosomes (dopamine), chromaffin cell membranes (NE), and platelet membranes (serotonin). Photoaffinity labeling of HeLa cell membranes which contain overexpressed dopamine and norepinephrine transporters will also be performed. Transporters which are labeled with these photoprobes will be identified by SDS-PAGE and pharmacologically characterized, as defined by the inhibition of labeling by specific monoamine uptake inhibitors (i.e., mazindol and GBR 12935 for the dopamine transporter, imipramine and fluoxetine for the serotonin transporter, and desipramine for the norepinephrine transporter). In the cases where two or more photoprobes (i.e., derivatives of cocaine, imipramine, and GBR 12935) label the same protein, peptide mapping will be performed to assess the identity of their binding sites. All photolabels will be characterized functionally as inhibitors of dopamine, norepinephrine, and serotonin transport using striatal synaptosomes, chromaffin cells, and platelets, respectively. HeLa cells which overexpress the dopamine and norepinephrine transporters will also be used in functional assays for cocaine photolabels. Cocaine affinity column resins will be synthesized. These resins will be chemically and functionally characterized and used towards purifying monoamine transporters from detergent extracts of HeLa membranes which contain overexpressed dopamine and norepinephrine transporters. A combination of biochemical techniques will be used for purification. Photolabeled peptides will be purified and sequenced to identify the cocaine binding site. The approaches outlined in this proposal will be used to characterize the molecular mechanism of action of cocaine towards a more rational approach in the effective treatment of cocaine abuse.
| Endres, C J; Swaminathan, S; DeJesus, O T et al. (1997) Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: implications for PET dopamine studies. Life Sci 60:2399-406 |
