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Volume 272, Number 41, Issue of October 10, 1997 pp. 26049-26055
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Peptide Mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3'-Iodo-4'-azidophenyl)propionyl]tetrabenazine Binding Sites for the Synaptic Vesicle Monoamine Transporter

(Received for publication, April 9, 1997, and in revised form, July 29, 1997)

Michael K. Sievert and Arnold E. Ruoho

From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 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 photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 µM tetrabenazine (TBZ), 10 µM 7-aminoketanserin, and 1 mM concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.


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Copyright © 1997 by the American Society for Biochemistry and Molecular Biology.