Abstract
Neuroactive steroids modulate the function of γ-aminobutyric acid type A (GABAA) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3α,5β)-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 GABAA receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABAA receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABAA 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 GABAA receptor function as evidenced by radioligand ([35S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABAA receptor function normally in VDAC-2-deficient fibroblasts transfected with α1β2γ2 GABAA receptor subunits. Finally, the neuroactive steroid pregnanolone [(3α,5β)-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 GABAA receptor modulation or anesthesia.
Footnotes
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This work was supported in part by National Institutes of Health Grants P01-GM47969 (to J.H.S., D.F.C., and A.S.E.), AA12952 (to S.J.M.), and R01 GM055713 (to W.J.C.), by a grant from the Klingenstein Foundation (to S.J.M.) and by the Alcoholic Beverage Medical Research Foundation (to G.A.).
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DOI: 10.1124/jpet.103.058123.
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ABBREVIATIONS: 6-AziP, (3α,5β)-6-azi-3-hydroxypregnan-20-one; VDAC, voltage-dependent anion channel; G418, geneticin; TBPS, t-butylbicyclophosphorothionate; ACN, (3α,5α)-3-hydroxyandrostan-20-carbonitrile; PAGE, polyacrylamide gel electrophoresis; CTβ, activation-related closing time; 3α5αP, allopregnanolone [(3α,5α)-3-hydroxypregnan-20-one]; 3α5βP, pregnanolone [(3α,5β)-3-hydroxypregnan-20-one].
- Received August 23, 2003.
- Accepted October 30, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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