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

Neuroactive steroids modulate the function of γ-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, (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 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 α₁β₂γ₂ GABA_A 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 GABA_A receptor modulation or anesthesia.