Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor
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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)
- ORCID
- Venue
- Molecular Pharmacology
- Publisher
- Elsevier BV
- Published
- 2012-09
- Volume
- 82
- Issue
- 3
- Pages
- 408-419
- ISSN
- 0026-895X (ISSN-L)
- PMC
- PMC3422701
- 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
- bronze
- doi.org
- 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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