Identification of drug binding sites on the vesicular monoamine transporter

The sequestration of neurotransmitters into presynaptic storage vesicles is an important part of the overall process of neurotransmission. The protein responsible for this sequestration is the vesicular monoamine transporter (VMAT). The cloning of the vesicular monoamine transporters has been a major advance in the general understanding of structure and function of these proteins; however, many questions still remain, such as the location of membrane-spanning domains and ligand binding domains. The technique of photoaffinity labeling has proven to be a useful approach for the identification of ligand binding sites. In this thesis, the synthesis and characterization of several photoaffinity labels for the vesicular monoamine transporter are presented, as well as a methodology for the expression and purification of large amounts of the vesicular monoamine transporter.; Three carrier-free radioiodinated photoaffinity labels for the vesicular monoamine transporter have been synthesized, 18-O- (N-(3{dollar}spprime{dollar}-iodo-4{dollar}spprime{dollar}-azidophenethyl) glycyl) methyl reserpate ( ({dollar}sp{lcub}125{rcub}{dollar}I) IAPEGlyMER), 2-N- ((3{dollar}spprime{dollar}-iodo-4{dollar}spprime{dollar}-azidophenyl)propionyl) tetrabenazine ( ({dollar}sp{lcub}125{rcub}{dollar}I) TBZ-AIPP), and 7-azido-8-iodoketanserin ( ({dollar}sp{lcub}125{rcub}{dollar}I) AZIK). These photoaffinity labels have been shown to specifically label VMAT from chromaffin granules. Photoincorporation of these labels into VMAT was protectable by both ligands and substrates of VMAT.; The rat synaptic vesicle monoamine transporter was cloned into the baculovirus DNA and expressed in insect cells. Baculovirus-expressed rVMAT2 exhibited the expected pharmacology of previously characterized VMAT's. A hexahistidine epitope was included at the C-terminus of the recombinant VMAT to aid in the purification. Baculovirus-expressed rVMAT2 could be purified in large quantities to greater than 95% homogeneity, features which helped in performing the protein chemistry of the transporter.; Using these tools, the binding site peptides for ({dollar}sp{lcub}125{rcub}{dollar}I) AZIK and ({dollar}sp{lcub}125{rcub}{dollar}I) TBZ-AIPP on rVMAT2 were identified. ({dollar}sp{lcub}125{rcub}{dollar}I) AZIK inserted into the N-terminus of rVMAT2, derivatizing lys20, the last amino acid prior to putative transmembrane domain 1 (TMD1). ({dollar}sp{lcub}125{rcub}{dollar}I) TBZ-AIPP inserted into a 24 amino acid fragment between putative TMD10 and TMD11. These data indicate that TMD1 and TMD10/11 of vesicular monoamine transporter interact with ({dollar}sp{lcub}125{rcub}{dollar}I) AZIK and ({dollar}sp{lcub}125{rcub}{dollar}I) TBZ-AIPP, respectively.