PEPTIDE-MAPPING OF THE [I-125] IODOAZIDOKETANSERIN AND [I-125] N-[(3'-IODO-4'-AZIDOPHENYL)PROPIONYL]TETRABENAZINE BINDING-SITES FOR THE SYNAPTIC VESICLE MONOAMINE TRANSPORTER

The full-length cDNA for the rat recombinant synaptic vesicle monoamin e transporter (rVMAT2) containing a COOH-terminal polyhistidine epitop e was engineered into baculovirus DNA for expression in Spodoptera fru giperda (Sf9) cells. Using this recombinant baculovirus and cultured S f9 cells, rVMAT2 has been expressed to high levels and purified to >95 % homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was ph otolabeled using [I-125]-7-azido-8-iodoketanserin ([I-125]AZIK) and -[ (3'-iodo-4'-azido-phenyl)propionyl]tetrabenazine ([I-125]TBZ-AIPP). Bo th [I-125]AZIK [I-125]TBZ-AIPP photoaffinity labeling of purified rVMA T2 were protectable by 10 mu M tetrabenazine (TBZ), 10 mu M 7-aminoket anserin, and 1 mM concentrations of the transporter substrates dopamin e, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl s ulfate-polyacrylamide gel electrophoresis, and NH2-terminal microseque nced. Radiosequencing of [I-125]AZIK-labeled rVMAT2 indicated derivati zation of Lys-20 in the NH2 terminus, just prior to putative transmemb rane domain 1 (TMD1). [I-125]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxt aposition of TMD1 and 10/11.