SELECTIVE ACTIONS OF CERTAIN NEUROACTIVE PREGNANEDIOLS AT THE GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR COMPLEX IN RAT-BRAIN

Certain endogenous pregnanediols (5 alpha-pregnan-3 alpha,20 alpha-dio l and 5 beta-pregnan-3 alpha,20 beta-diol) were observed to have limit ed efficacy as allosteric modulators of t-[S-35]butylbicyclophosphorot hionate ([S-35]TBPS) and [H-3]flunitrazepam binding to sites on the ga mma-aminobutyric acid (GABA)(A)-receptor complex in rat brain. In cont rast, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 beta-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) have full effic acy. Moreover, 3 alpha,5 beta-P but not 3 alpha,5 alpha-P recognizes h igh (nanomolar) and low (micromolar) affinity neuroactive steroid site s in these allosteric modulatory assays. The concentration-response cu rve for 3 alpha,5 alpha-P modulation of [S-35]TBPS binding was shifted rightward in the presence of these pregnanediols and GABA. The maximu m shift produced by these pregnanediols never exceeded the concentrati on-response curve obtained with 3 alpha,5 alpha-P alone in the absence of GABA. Additionally, neither 5 alpha-pregnan-3 alpha,20 alpha-diol nor 5 beta-pregnan-3 alpha,20 beta-diol had any effect on the site rec ognized by 3 alpha,5 alpha-P in the absence of GABA. The difference in the affinities of the two apparent sites (29 nM versus 152 nM in the presence and absence of GABA, respectively) recognized by 3 alpha,5 al pha-P is only similar to 5-fold. In contrast, the difference between t he high (30 nM) and low (7 mu M) affinity sites discriminated by 3 alp ha,5 beta-P is >200-fold. Thus, the selective interaction between the high affinity site recognized by 3 alpha,5 beta-P and these pregnanedi ols can be clearly observed. A saturating concentration of 5 beta-preg nan-3 alpha,20 beta-diol selectively eliminated the high affinity comp onent recognized by 3 alpha,5 beta-P, whereas 5 alpha-pregnan-3 alpha, 20 alpha-diol did not completely abolish the high affinity site. 5 alp ha-Pregnan-3 alpha,20 alpha-diol recognized only a portion of the high affinity sites discriminated by 3 alpha,5 beta-P, relative to 5 beta- pregnan-3 alpha,20 beta-diol, whereas the two pregnanediols recognized a similar population of sites mediating 3 alpha,5 alpha-P inhibition of [S-35]TBPS binding. Collectively, these studies provide evidence th at the limited efficacy of certain pregnanediols as allosteric modulat ors of [S-35]TBPS binding may be explained in part by selectivity for the high affinity site recognized by 3 alpha,5 beta-P. Data collected from 5 alpha-pregnan-3 alpha,20 alpha-diol modulation of [S-35]TBPS bi nding to recombinantly expressed receptors suggest that the subunit co mposition of the GABA(A) receptor complex may contribute to pregnanedi ol selectivity, The relative contributions of neuroactive steroid rece ptor subtypes and/or different affinity states of the same receptor to the apparent receptor heterogeneity observed remain to be determined.