Neuroactive steroid interactions with voltage-dependent anion channels: lack of relationship to GABA(A) receptor modulation and anesthesia.
Neuroactive steroids modulate the function of gamma-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, (3alpha,5beta)-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 [(3)H]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 ([(35)S] 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 alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-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.
Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ramin
R
Darbandi-Tonkabon
Brad D
BD
Manion
William R
WR
Hastings
William J
WJ
Craigen
Gustav
G
Akk
John R
JR
Bracamontes
Yejun
Y
He
Tatiana V
TV
Sheiko
Joseph H
JH
Steinbach
Steven J
SJ
Mennerick
Douglas F
DF
Covey
Alex S
AS
Evers
D000758:Anesthesia / Q000662:veterinary; D000818:Animals; D001388:Aziridines / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D004594:Electrophysiology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D007473:Ion Channels / Q000172:deficiency / Q000378:metabolism; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000172:deficiency / Q000378:metabolism; D010777:Photochemistry; D018272:Porins / Q000172:deficiency / Q000378:metabolism*; D011280:Pregnanolone / Q000031:analogs & derivatives* / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology*; D013462:Sulfur Radioisotopes; D050995:Voltage-Dependent Anion Channel 1; D050996:Voltage-Dependent Anion Channel 2; D050994:Voltage-Dependent Anion Channels
C475062:6-azipregnanolone; D001388:Aziridines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007473:Ion Channels; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D018272:Porins; D011963:Receptors, GABA-A; D013256:Steroids; D013462:Sulfur Radioisotopes; C494763:Vdac1 protein, mouse; C494766:Vdac2 protein, mouse; C480078:Vdac3 protein, mouse; D050996:Voltage-Dependent Anion Channel 2; D050994:Voltage-Dependent Anion Channels; C037476:tert-butylbicyclophosphorothionate; D011280:Pregnanolone; D050995:Voltage-Dependent Anion Channel 1