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 GABA A 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 [(3α5β)-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.
|Number of pages||10|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Feb 2004|