QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.107.034439v1 72/6/1567    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gopalakrishnan, A. Articles by Ruoho, A. E. Search for Related Content PubMed PubMed Citation Articles by Gopalakrishnan, A. Articles by Ruoho, A. E.

Identification of the Substrate Binding Region of Vesicular Monoamine Transporter-2 (VMAT-2) Using Iodoaminoflisopolol as a Novel Photoprobe

Department of Pharmacology, University of Wisconsin Madison, Madison, Wisconsin

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.

Address correspondence to: Dr. Arnold E. Ruoho, Department of Pharmacology, 4775 SMI, 1300 University Avenue, Madison WI 53705. E-mail: aeruoho{at}wisc.edu

This article has been cited by other articles:

				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]