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First published on August 31, 2007; DOI: 10.1124/mol.107.034439

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Received for publication February 15, 2007.
Revised August 14, 2007.
Accepted for publication August 30, 2007.

IDENTIFICATION OF THE SUBSTRATE BINDING REGION OF VMAT-2 USING IODOAMINOFLISOPOLOL AS A NOVEL PHOTOPROBE

Anupama Gopalakrishnan 1, Michael K Sievert 1, Arnold E. Ruoho 1*

1 University of Wisconsin Medical School

* Address correspondence to: E-mail: aeruoho{at}wisc.edu

Abstract

Monoamines, such as serotonin, dopamine and norepinephrine, are sequestered into synaptic vesicles by specific transporters (VMAT2), utilizing 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 inhibition of [3H] 5HT uptake 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 classical compounds such as [3H]5HT and [3H]TBZOH by incubating the compound with the vesicles, followed by washes with isotonic and hypotonic solutions. Using this method, we characterize IAmF as a novel VMAT2 substrate. VMAT2 containing Sf9 vesicles showed 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 TMs 6 and 7, followed by thrombin digestion retained photolabel in a 22KDa 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 VMAT2 molecule.


Key words: Adrenergic, Serotonin, Biogenic Amine, Receptor binding studies


This article has been cited by other articles:


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Y. Adam, R. H. Edwards, and S. Schuldiner
Expression and function of the rat vesicular monoamine transporter 2
Am J Physiol Cell Physiol, April 1, 2008; 294(4): C1004 - C1011.
[Abstract] [Full Text] [PDF]


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