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

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

Zi-Wei Chen, Brad Manion, R. Reid Townsend, David E. Reichert, Douglas F. Covey, Joe Henry Steinbach, Werner Sieghart, Karoline Fuchs and Alex S. Evers
Molecular Pharmacology September 2012, 82 (3) 408-419; DOI: https://doi.org/10.1124/mol.112.078410
Zi-Wei Chen
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brad Manion
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
R. Reid Townsend
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David E. Reichert
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Douglas F. Covey
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joe Henry Steinbach
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Werner Sieghart
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Karoline Fuchs
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alex S. Evers
Departments of Anesthesiology (Z.-W.C., B.M., J.H.S., A.S.E.), Internal Medicine (R.R.T., A.S.E.), Cell Biology and Physiology (R.R.T.), and Developmental Biology (D.F.C., A.S.E.) and Mallinckrodt Institute of Radiology (D.E.R.), Washington University in St. Louis, St. Louis, Missouri; and Department of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S., K.F.)
  • 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 + SI
  • PDF
Loading

Abstract

Accumulated evidence suggests that neurosteroids modulate GABAA receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His8-β3 homomeric GABAA receptors. 6-AziP inhibited 35S-labeled t-butylbicyclophosphorothionate binding to the His8-β3 homomeric GABAA receptors in a concentration-dependent manner (IC50 = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [3H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His8-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [3H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [3H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His8-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant PO1-GM47969] (to D.F.C., J.H.S., A.S.E.), [Grant 8P41-GM103422-35] (to R.R.T); the National Institutes of Health National Center for Research Resources [Grant 5P41RR000954-35] (to R.R.T), the Austrian Ministry of Science and Research; and the European Union Seventh Framework Program [Grant HEALTH-F4-2008-202088] (Neurocypres) (to W.S.).

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

    http://dx.doi.org/10.1124/mol.112.078410.

  • ABBREVIATIONS:

    TM
    transmembrane region
    LC
    liquid chromatography
    MS
    mass spectrometry
    TMD
    transmembrane domain
    FA
    formic acid
    PGC
    porous graphitic carbon
    ACN
    acetonitrile
    PAGE
    polyacrylamide gel electrophoresis
    SPE
    solid-phase extraction
    MS1
    precursor-ion mass spectrometry
    MS2
    product-ion mass spectrometry
    LTQ
    linear quadrupole ion trap
    DMSA
    directed product-ion spectral acquisition
    XIC
    extracted ion chromatogram
    VDAC
    voltage-dependent anion channel
    PDB
    Protein Data Bank
    Glu-Cl
    glutamate-gated chloride channel
    6-AziP
    6-azi-pregnanolone
    TBPS
    t-butyl-bicyclophosphorothionate
    E-64
    N-[N-(l-3-trans-carboxyirane-2-carbonyl)-l-leucyl]-agmatine
    TSA
    transformed human embryonic kidney.

  • Received February 20, 2012.
  • Accepted May 30, 2012.
  • Copyright © 2012 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: 82 (3)
Molecular Pharmacology
Vol. 82, Issue 3
1 Sep 2012
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • 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.
Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor
(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

6-AziP Photolabels TM3 of β3 Homomeric GABAA Receptors

Zi-Wei Chen, Brad Manion, R. Reid Townsend, David E. Reichert, Douglas F. Covey, Joe Henry Steinbach, Werner Sieghart, Karoline Fuchs and Alex S. Evers
Molecular Pharmacology September 1, 2012, 82 (3) 408-419; DOI: https://doi.org/10.1124/mol.112.078410

Citation Manager Formats

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

Share
Research ArticleArticle

6-AziP Photolabels TM3 of β3 Homomeric GABAA Receptors

Zi-Wei Chen, Brad Manion, R. Reid Townsend, David E. Reichert, Douglas F. Covey, Joe Henry Steinbach, Werner Sieghart, Karoline Fuchs and Alex S. Evers
Molecular Pharmacology September 1, 2012, 82 (3) 408-419; DOI: https://doi.org/10.1124/mol.112.078410
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Authorship Contributions
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Cysteine151 in Keap1 Drives CDDO-Me Pharmacodynamic Action
  • Allosteric Modulation of Metabotropic Glutamate Receptor 1
  • Mechanism of Selective Action of Paraherquamide A
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