Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Molecular Pharmacology
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • My Cart
Molecular Pharmacology

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit molpharm on Facebook
  • Follow molpharm on Twitter
  • Follow molpharm on LinkedIn
Research ArticleArticle

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

Anupama Gopalakrishnan, Michael Sievert and Arnold E. Ruoho
Molecular Pharmacology December 2007, 72 (6) 1567-1575; DOI: https://doi.org/10.1124/mol.107.034439
Anupama Gopalakrishnan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Sievert
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Arnold E. Ruoho
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Abstract

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.

Footnotes

  • This work was supported by National Institutes of Health grant R01-NS033650 (to A.E.R.).

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

  • doi:10.1124/mol.107.034439.

  • ABBREVIATIONS: VMAT2, vesicular monoamine transporter-2; IAmF, iodoaminoflisopolol; AZIK, 7-iodo-8-azido ketanserin; TBZ, tetrabenazine; CCCP, carbonylcyanide-m-chlorophenylhydrazone; TBZOH, dihydrotetrabenazine; 5-HT, 5-hydroxytryptamine; β2AR, β2-adrenergic receptor; SH, sucrose-HEPES; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; TM, transmembrane.

    • Received February 14, 2007.
    • Accepted August 30, 2007.
  • The American Society for Pharmacology and Experimental Therapeutics
View Full Text

MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years. 

Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page. 

 

  • Click here for information on institutional subscriptions.
  • Click here for information on individual ASPET membership.

 

Log in using your username and password

Forgot your user name or password?

Purchase access

You may purchase access to this article. This will require you to create an account if you don't already have one.
PreviousNext
Back to top

In this issue

Molecular Pharmacology: 72 (6)
Molecular Pharmacology
Vol. 72, Issue 6
1 Dec 2007
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Back Matter (PDF)
  • Editorial Board (PDF)
  • Front Matter (PDF)
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Molecular Pharmacology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Identification of the Substrate Binding Region of Vesicular Monoamine Transporter-2 (VMAT-2) Using Iodoaminoflisopolol as a Novel Photoprobe
(Your Name) has forwarded a page to you from Molecular Pharmacology
(Your Name) thought you would be interested in this article in Molecular Pharmacology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

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

Anupama Gopalakrishnan, Michael Sievert and Arnold E. Ruoho
Molecular Pharmacology December 1, 2007, 72 (6) 1567-1575; DOI: https://doi.org/10.1124/mol.107.034439

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

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

Anupama Gopalakrishnan, Michael Sievert and Arnold E. Ruoho
Molecular Pharmacology December 1, 2007, 72 (6) 1567-1575; DOI: https://doi.org/10.1124/mol.107.034439
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Mechanism of the selective action of paraherquamide A
  • Use-Dependent Relief of A-887826 Inhibition
  • Benzbromarone Relaxes Airway Smooth Muscle via BK Activation
Show more Articles

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About Molecular Pharmacology
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Drug Metabolism and Disposition
  • Journal of Pharmacology and Experimental Therapeutics
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-0111 (Online)

Copyright © 2023 by the American Society for Pharmacology and Experimental Therapeutics