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) recognitio n site labeled by the antagonist ligand [H-3]SR 95531 was evaluated in rat brain cortical membranes. The first type is the GABA site antagon ist RU 5135, which potently (IC50 7 nM) but also incompletely (I-max 8 2%) displaced [H-3]SR 95531. RU 5135 probably binds only to high affin ity [H-3]SR 95531 sites recognized by GABA and unlabelled SR 95531. Th e second type are the neuroactive steroids which act as positive allos teric modulators, including 3 alpha-hydroxy-5 beta-pregnan-20-one (3 a lpha,5 beta-P) and 5 beta-tetrahydrodeoxycorticosterone (5 beta-THDOC) , which inhibited [H-3]SR 95531 binding with limited efficacy (IC50 46 0 nM and 1.4 mu M, I-max 41 and 31%, respectively). In contrast, 3 alp ha-hydroxy-5 alpha-pregnan-2D-one (3 alpha,5 beta-P) was inactive. The third type are the neurosteroids acting as negative allosteric modula tors, such as pregnenolone sulfate, which inhibited [H-3]SR 95531 bind ing with limited efficacy (IC50 10 mu M, I-max 23%). In the presence o f a saturating concentration of pregnenolone sulfate, 3 alpha,5 beta-P further inhibited [H-3]SR 95531 binding suggesting that these two ste roids act through different sites or, possibly, at different populatio ns of GABA(A) receptors. The allosteric modulation was selective for s teroids 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 al pha,5 alpha-P, pregnenolone sulfate, GABA, benzodiazepines, and barbit urates.