ArticleMolecular pharmacology2012
Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.
Article in Molecular pharmacology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 54 papers.
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Who cites it
54 citing papers in PubMed.
- New directions in neurosteroid therapeutics in neuropsychiatry.Neuroscience and biobehavioral reviews · 2025Review
- Direct measurements of neurosteroid binding to specific sites on GABABritish journal of pharmacology · 2024Article
- A bupropion modulatory site in the Gloeobacter violaceus ligand-gated ion channel.Biophysical journal · 2024Article
- Neurosteroids and their potential as a safer class of general anesthetics.Journal of anesthesia · 2024Review
- Phenols and GABAFrontiers in pharmacology · 2024Review
- Structural insights into opposing actions of neurosteroids on GABANature communications · 2023Article
- Neurosteroid enantiomers as potentially novel neurotherapeutics.Neuroscience and biobehavioral reviews · 2023Review
- The Mechanism of Enantioselective Neurosteroid Actions on GABABiomolecules · 2023Article
- Neurosteroid Modulation of GABACurrent neuropharmacology · 2022Article
- Article
- Analysis of Modulation of theMolecular pharmacology · 2020Article
- The Biology of General Anesthesia from Paramecium to Primate.Current biology : CB · 2019Review
- Neurosteroids as novel antidepressants and anxiolytics: GABA-A receptors and beyond.Neurobiology of stress · 2019Article
- The molecular determinants of neurosteroid binding in the GABA(A) receptor.The Journal of steroid biochemistry and molecular biology · 2019Article
- Multiple functional neurosteroid binding sites on GABAA receptors.PLoS biology · 2019Article
- Common binding sites for cholesterol and neurosteroids on a pentameric ligand-gated ion channel.Biochimica et biophysica acta. Molecular and cell biology of lipids · 2019Article
- Neurosteroid regulation of GABABrain research · 2019Review
- Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors.European journal of medicinal chemistry · 2019Article
- Comparison of αβδ and αβγ GABAPharmacological research · 2018Review
- A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7.Journal of lipid research · 2018Article
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Authors and funding
9 authors.
Funding
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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