Identification of the substrate binding region of vesicular monoamine transporter-2 (VMAT-2) using iodoaminoflisopolol as a novel photoprobe.
Monoamines, such as serotonin, dopamine, and norepinephrine, are sequestered into synaptic vesicles by specific transporters (vesicular monoamine transporter-2; VMAT2) using energy from an electrochemical proton gradient across the vesicle membranes. Based on our previous studies using photoaffinity-labeling techniques in characterizing the VMAT2-specific ligands ketanserin and tetrabenazine, this study describes the synthesis and characterization of a fluorenone-based compound, iodoaminoflisopolol (IAmF), as a photoprobe to identify the substrate binding site(s) of VMAT2. Using vesicles prepared from rat VMAT2 containing recombinant baculovirus-infected Sf9 cells, we show the inhibition of [3H]5-hydroxytryptamine (5-HT) uptake and [3H]dihydrotetrabenazine (TBZOH) binding by aminoflisopolol and iodoaminoflisopolol. The interaction of [125I]IAmF with VMAT2 is highly dependent on the presence of ATP and an intact proton gradient. We report a simple and novel method to distinguish between a ligand and substrate using classic compounds such as [3H]5-HT and [3H]TBZOH by incubating the compound with the vesicles followed by washes with isotonic and hypotonic solutions. Using this method, we confirm the characterization of IAmF as a novel VMAT2 substrate. Sf9 vesicles expressing VMAT2 show reserpine- and tetrabenazine-protectable photolabeling by [125I]IAmF. [125I]IAmF photolabeling of recombinant VMAT2, expressed in SH-SY5Y cells with an engineered thrombin site between transmembranes 6 and 7, followed by thrombin digestion, retained photolabel in a 22-kDa fragment, indicating that iodoaminoflisopolol binds to the C-terminal half of the VMAT2 molecule. Thus, IAmF possesses a unique combination of VMAT2 substrate properties and a photoprobe and is, therefore, useful to identify the substrate binding site of the vesicular transporter.
Gopalakrishnan
Anupama
A
Department of Pharmacology, University of Wisconsin Madison, Madison WI 53705, USA.
Sievert
Michael
M
Ruoho
Arnold E
AE
D058672:Adrenergic beta-2 Receptor Antagonists; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D005449:Fluorenes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D015335:Molecular Probes / Q000032:analysis / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D050493:Vesicular Monoamine Transport Proteins / Q000032:analysis / Q000378:metabolism*
C415883:4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone; D058672:Adrenergic beta-2 Receptor Antagonists; D005449:Fluorenes; D015335:Molecular Probes; D018343:Receptors, Adrenergic, beta-2; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins