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Photoaffinity Labeling of a Neuroactive Steroid Binding Protein in Rat Brain
Ramin Darbandi-Tonkabon, M.D.; Randy Hastings, B.S.; Chun-Min Zeng, Ph.D.; Douglas F. Covey, Ph.D.; Alex S. Evers, M.D.
Departments of Anesthesiology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri
INTRODUCTION: It is generally thought that neuroactive steroids produce anesthesia by modulating the function of GABA-A receptors, and that this modulation is mediated by specific neuroactive steroid recognition sites on proteins-presumably the GABA-A receptor. In this study we have used a novel photoaffinity labeling reagent, [3H]Photopregnanalone to identify neuroactive steroid binding sites in rat brain membranes.

METHODS: [3H]Photopregnanalone was made by incorporating a diazirene moiety into the neuroactive steroid, pregnanalone (3α-hydroxy-5β-pregnan-20-one). Previously described methods were used to assess the biological activity of Photopregnanalone.1 For photolabeling studies, rat brain membranes (P2 fraction) were incubated with [3H]Photopregnanalone (100 nM- 10 μM) and exposed to light (> 315 nm) for 3 min. Membranes were collected by centrifugation and proteins were separated by SDS-PAGE. Gels were analyzed by scintillation spectrometry of gel slices or by autoradiography. In immunoprecipitation studies, precipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed using antisera (bd17) directed to the β-subunits of the GABA-A receptor.

RESULTS: Photopregnanalone inhibited binding of [35S]TBPS to rat brain membranes and potentiated GABA-elicited currents in Xenopus oocytes expressing GABA-A receptor subunits (α1β2γ2). Photopregnanalone was rapidly photolyzed by exposure to light at 350 nm, and covalently photolabeled model substrates. [3H]Photopregnanalone produced concentration-dependent photolabeling of a 35-kDa protein in rat brain membranes, with half-maximal labeling observed at 3μM. Photolabeling of the 35-kDa protein was largely prevented when incubations were performed in the presence of an excess (30 μM) of pregnanalone. The 35-kDa was also photolabeled by [3H]Photopregnanalone in membranes prepared from cells (HEK, QT-6) not expressing GABA-A receptors; this indicated that the 35-kDa protein is widely expressed and is unlikely to be a GABA-A receptor subunit fragment. The photolabeled 35-kDa protein was isolated by 2-dimensional PAGE. Anti-sera generated to the 35-kDa protein were shown to immunoprecipitate [3H]Photopregnanalone-labeled 35-kDa protein and to co-immunoprecipitate GABA-A receptors, as evidenced by bd17 staining of proteins corresponding to the β2 and β3 subunits of the GABA-A receptor.

CONCLUSIONS: This study demonstrates the existence of a specific neuroactive steroid binding protein in rat brain. This protein is ubiquitously expressed but is physically associated with GABA-A receptors in rat brain. These results suggest the possibility that neuroactive steroids produce their effects by binding to a GABA-A receptor-accessory protein rather than to the GABA-A receptor itself. Identification of the 35-kDa protein and delineation of its functional interaction with the GABA-A receptor will be necessary to further explore this hypothesis.

REFERENCES: 1) Mennerick S. Zeng CM et. al., Mol Pharmacol, 60:732-41, (2001)

Anesthesiology 2002; 96: A814