Steroid inhibition of [H-3]SR 95531 binding to the GABA(A) recognition site

The interaction of three types of steroids with the GABA(A) recognition site labeled by the antagonist ligand [H-3]SR 95531 was evaluated in rat brain cortical membranes. The first type is the GABA site antagonist RU 5135, which potently (IC50 7 nM) but also incompletely (I-max 82%) displaced [H-3]SR 95531. RU 5135 probably binds only to high affinity [H-3]SR 95531 sites recognized by GABA and unlabelled SR 95531. The second type are the neuroactive steroids which act as positive allosteric modulators, including 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) and 5 beta-tetrahydrodeoxycorticosterone (5 beta-THDOC), which inhibited [H-3]SR 95531 binding with limited efficacy (IC50 460 nM and 1.4 mu M, I-max 41 and 31%, respectively). In contrast, 3 alpha-hydroxy-5 alpha-pregnan-2D-one (3 alpha,5 beta-P) was inactive. The third type are the neurosteroids acting as negative allosteric modulators, such as pregnenolone sulfate, which inhibited [H-3]SR 95531 binding with limited efficacy (IC50 10 mu M, I-max 23%). In the presence of a saturating concentration of pregnenolone sulfate, 3 alpha,5 beta-P further inhibited [H-3]SR 95531 binding suggesting that these two steroids act through different sites or, possibly, at different populations of GABA(A) receptors. The allosteric modulation was selective for steroids since benzodiazepines and barbiturates were inactive up to 100 mu M. Taken together, these data suggest that 3 alpha,5 beta-P and 5 beta-THDOC modulate [H-3]SR 95531 binding by interacting with a unique site on the GABA(A) receptor complex distinct from the sites for 3 alpha,5 alpha-P, pregnenolone sulfate, GABA, benzodiazepines, and barbiturates.