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Originally published In Press as doi:10.1074/jbc.M213168200 on January 30, 2003

J. Biol. Chem., Vol. 278, Issue 15, 13196-13206, April 11, 2003
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Photoaffinity Labeling with a Neuroactive Steroid Analogue
6-AZI-PREGNANOLONE LABELS VOLTAGE-DEPENDENT ANION CHANNEL-1 IN RAT BRAIN*

Ramin Darbandi-TonkabonDagger , William R. HastingsDagger , Chun-Min Zeng§, Gustav AkkDagger , Brad D. ManionDagger , John R. BracamontesDagger , Joseph H. SteinbachDagger , Steven J. Mennerick, Douglas F. Covey§, and Alex S. EversDagger §||

From the Departments of Dagger  Anesthesiology, § Molecular Biology and Pharmacology, and  Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110

Neuroactive steroids modulate the function of gamma -aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3alpha ,5beta -6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (alpha 1beta 2gamma 2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ~35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 µM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The beta 2 and beta 3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1.

* This work was supported in part by National Institutes of Health Grants PO1-GM47969 (to J. H. S., D. F. C., and A. S. E.) and AA12952 (to S. J. M.) and by a grant from the Klingenstein Foundation (to S. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8054, St. Louis, MO 63110. Tel.: 314-454-8702; Fax: 314-454-5572; E-mail: eversa@notes.wustl.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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