Cited by 73Neuroscience and Neuropharmacology ResearchReceptor Mechanisms and SignalingNicotinic Acetylcholine Receptors Study

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

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Chen, Zi-Wei, Manion, Brad, Townsend, R. Reid, Reichert, David E., Covey, Douglas F., Steinbach, Joe Henry, Sieghart, Werner, Fuchs, Karoline +1 more·2012·Molecular Pharmacology

Abstract

Accumulated evidence suggests that neurosteroids modulate GABA(A) 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, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]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 His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-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.

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  • Clinically relevant low doses of all volatile anesthetics potentiate GABAA signaling.

    “Clinically relevant, low doses of all volatile anesthetics have been shown to potentiate GABAA signaling [34]”

    From The Biology of General Anesthesia from Paramecium to Primate (2019) · cites this paper as [34]

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Authors
Chen, Z.-W., Manion, B., Townsend, R. R., Reichert, D. E., Covey, D. F., Steinbach, J. H., Sieghart, W., Fuchs, K., & Evers, A. S.
Affiliations
Washington University in St. Louis (US)
Venue
Molecular Pharmacology
Publisher
Elsevier BV
Published
2012-09
Volume
82
Issue
3
Pages
408-419
ISSN
0026-895X (ISSN-L)
OpenAlex
W2131961672
Cited by
72
References
42
Citations by year
2025: 3 · 2024: 5 · 2023: 3 · 2021: 4 · 2020: 1 · 2019: 4 · 2018: 10 · 2017: 12 · 2016: 8 · 2015: 5 · 2014: 8 · 2013: 5 · 2012: 4
FWCI
2.90
Type
journal article
Language
en
Open access
Locations
  • Molecular Pharmacology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • European Commission (202088)
  • National Institutes of Health (5p41rr000954-34, GM47969, GM103422)
  • National Institute of General Medical Sciences (GM103422, GM-47969, P01GM047969, P01GM47969, P41GM103422)
  • National Center for Research Resources (P41-RR000954, 5P41RR000954-35)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nicotinic Acetylcholine Receptors Study
Keywords
neurosteroids, GABAA receptor, beta3 subunit, third transmembrane domain, photolabeling, stereoselective binding, ligand-gated ion channels
MeSH terms
Amino Acid Sequence, Animals, Aziridines, Binding Sites, Brain, Cells, Cultured, Humans, Models, Molecular, Molecular Sequence Data, Pregnanolone, Receptors, GABA-A, Immunoblotting, Protein Structure, Tertiary, Neurotransmitter Agents, Photoaffinity Labels, Protein Subunits, Rats

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