Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain.
Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.
Darbandi-Tonkabon
Ramin
R
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Hastings
William R
WR
Zeng
Chun-Min
CM
Akk
Gustav
G
Manion
Brad D
BD
Bracamontes
John R
JR
Steinbach
Joseph H
JH
Mennerick
Steven J
SJ
Covey
Douglas F
DF
Evers
Alex S
AS
D000345:Affinity Labels / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001388:Aziridines / Q000493:pharmacokinetics / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009118:Muscimol / Q000493:pharmacokinetics; D009865:Oocytes / Q000502:physiology; D018272:Porins / Q000032:analysis / Q000502:physiology*; D011280:Pregnanolone / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013256:Steroids / Q000493:pharmacokinetics; D013687:Telencephalon / Q000378:metabolism*; D014162:Transfection; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
C475062:6-azipregnanolone; D000345:Affinity Labels; D001388:Aziridines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018272:Porins; D011994:Recombinant Proteins; D013256:Steroids; C494764:Vdac1 protein, rat; D050994:Voltage-Dependent Anion Channels; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; C037476:tert-butylbicyclophosphorothionate; D011280:Pregnanolone; D050995:Voltage-Dependent Anion Channel 1