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CELLULAR AND MOLECULAR

Neuroactive Steroid Interactions with Voltage-Dependent Anion Channels: Lack of Relationship to GABA_A Receptor Modulation and Anesthesia

Departments of Anesthesiology (R.D., B.D.M., W.R.H., G.A., J.R.B., J.H.S., A.S.E.), Molecular Biology and Pharmacology (D.F.C., A.S.E.), and Psychiatry (Y.H., S.J.M.), Washington University School of Medicine, St. Louis, Missouri; and Departments of Pediatrics and Genetics (T.V.S., W.J.C.), Baylor College of Medicine, Houston, Texas

Neuroactive steroids modulate the function of -aminobutyric acid type A (GABA_A) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3,5)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA_A receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA_A receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA_A receptor function and anesthesia. Photolabeling of 35-kDa protein with [³H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA_A receptor function as evidenced by radioligand ([³⁵S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA_A receptor function normally in VDAC-2-deficient fibroblasts transfected with ₁₂₂ GABA_A receptor subunits. Finally, the neuroactive steroid pregnanolone [(3,5)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA_A receptor modulation or anesthesia.

Address correspondence to: Dr. Alex S. Evers, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8054, St. Louis, MO 63110. E-mail: eversa{at}notes.wustl.edu

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