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 Ni 2؉ -affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using The accumulation of amines into storage granules in neurons, into chromaffin granules in the adrenal gland, and into granules of peripheral cells such as mast cells, occurs via a monoamine transporter found in the vesicle membrane which is inhibitable by reserpine, tetrabenazine (TBZ), 1 and ketanserin (for review, see Refs. 1, 2-8). The energy for amine transport is derived from a proton gradient, generated by ATP hydrolysis, and a membrane potential (8 -10). Two protons are released from the storage vesicle in exchange for one substrate molecule transported to the inside (11, 12). The proton gradient is coupled, by an unknown mechanism, to the transport of biogenic amines into the synaptic vesicle against a steep concentration gradient. The monoamine transporter transports numerous substrates across the vesicle membrane, including dopamine, norepinephrine, epinephrine, serotonin, histamine, tyramine, meta-iodobenzylguanidine, and the neurotoxin Nmethyl-4-pyridinium (13-16).The monoamine transporter was originally cloned from both rat PC12 cells and rat brain (17). The rat chromaffin granule transporter (VMAT1) contains 521 amino acids, and the rat synaptic vesicle transporter (VMAT2) contains 515 amino acids. Analysis by the method of hydrophobic moments implicates 12 transmembrane helices, which is a characteristic of other known transport proteins (18,19). The primary sequence of both transporters predicts three potential sites of N-linked glycosylation in the large luminal loop between transmembrane helices 1 and 2. The vesicular transporters show some similarity to the bacterial multidrug resistance transporter, which is also inhibitable by reserpine (20).A transporter with a sequence identical to that of the rat VMAT2 was expression cloned from rat CV-1 cells (21). The transporter activity was assayed by [ 3 H]serotonin uptake into permeabilized cells. In addition, VMAT2 has been cloned from a human brainstem cDNA library and from bovine chromaffin cell cDNA using probes derived from the rat VMAT2 (22)(23)(24)(25). The rat VMAT2 is highly similar to these VMATs; it shares 92 and 88% identity with human and bovine VMAT2, respectively. VMAT2 from rat, human, and bovine has similar pharmacological properties (26 -29).A major obstacle in studying the vesicle monoamine transporters at a molecular level has been the inability to obtain large amounts of purified transporter. In an earlier study, we reported the expression and purification of a recombinant synaptic vesicle monoamine transporter (rVMAT2) using the ...

Vesicular monoamine transporter 2: Role as a novel target for drug development

“…131 Photoaffi nity labeling of purifi ed rat VMAT2 indicates that TMD1 and TMD10/11 are possibly juxtaposed and may interact in a functionally signifi cant manner. 132 …”

Mutagenesis and Derivatization of Human Vesicle Monoamine Transporter 2 (VMAT2) Cysteines Identifies Transporter Domains Involved in Tetrabenazine Binding and Substrate Transport

“…Cys 439 is positioned in a hydrophobic environment (as predicted for the tetrabenazine binding site; Ref. 33), in the center of four mutations previously found to affect tetrabenazine binding (15,17,18), on a peptide that is photolabeled by a tetrabenazine photolabel (21), and on a transporter domain involved in TBZ binding identified from rat (16) and human (19) VMAT1/VMAT2 chimera studies. The cysteine derivatization and protection experiments described in this report support these earlier findings and significantly strengthen them against argument of the inherent uncertainties in interpreting site-directed mutagenesis data, or the possibility of photolabel insertion at a site somewhat distant from pharmacophore binding.…”

“…These studies have suggested important residues that may contribute to ligand binding and monoamine transport. In addition, two significant observations that have contributed VMAT2 structural information are the discovery that a lysine in predicted TM 2 and an aspartate in predicted TM 11 interact functionally as an ion pair (20), and the identification in rat VMAT2 of peptide domains near or at the ketanserin and tetrabenazine binding sites, and an amino acid residue (Lys 20 ) near or at the ketanserin binding site, from photoaffinity labeling studies performed in our laboratory (7,21). Although the reasonable inferences from molecular biology and the biochemical structural information from photoaffinity labeling have provided significant and novel information, a more general approach that can be applied to obtain structural information from the entire molecule and to map ligand binding sites has been sought.…”

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

“…Our previous studies have successfully used photoprobes to identify specific binding regions of interacting proteins and drug interaction with receptors (Sievert and Ruoho, 1997;Wu et al, 2001;Sievert et al, 2002;Guo et al, 2005Guo et al, , 2006. In a previous study, in an attempt to develop compounds capable of directly probing the catechol binding region of the ␤ 2 -adrenergic receptor (␤ 2 AR), our laboratory synthesized novel benzophenone-and fluorenone-based ␤ 2 AR antagonists as photoaffinity probes (Wu and Ruoho, 2000;Wu et al, 2001).…”

“…Using photoprobes, previous studies from our laboratory identified the ketanserin and tetrabenazine (TBZ) binding regions of VMAT2 (Sievert and Ruoho, 1997). Analyses of the binding site peptides showed that although the ketanserin photoprobe […”