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
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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.
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
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