title: Peptide mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine binding sites for the synaptic vesicle monoamine transporter.
abstract: 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 microM tetrabenazine (TBZ), 10 microM 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.
authors.affiliation: Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Sievert
authors.forename: A E
authors.initials: AE
authors.lastname: Ruoho
mesh_terms: D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D007457:Iodine Radioisotopes; D007650:Ketanserin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism*; D013747:Tetrabenazine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D050492:Vesicular Biogenic Amine Transport Proteins
chemical_list: C108672:2-N-((3'-iodo-4'-azidophenyl)propionyl)tetrabenazine; D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; C067115:azidoiodoketanserin; D007650:Ketanserin; D013747:Tetrabenazine