Selective actions of certain neuroactive pregnanediols at the gamma-aminobutyric acid type A receptor complex in rat brain
McCauley, L.D.; Liu, V.; Chen, J.S.; Hawkinson, J.E.; Lan, N.C.; Gee, K.W.
Molecular Pharmacology 47(2): 354-362
1995
ISSN/ISBN: 0026-895X PMID: 7870044 Document Number: 445152
Certain endogenous pregnanediols (5-alpha-pregnan-3-alpha,20-alpha-diol and 5-beta-pregnan-3-alpha,20-beta-diol) were observed to have limited efficacy as allosteric modulators of t-(35S)butylbicyclophosphorothionate ((35S)TBPS) and (3H)flunitrazepam binding to sites on the gamma-aminobutyric acid (GABA)-A receptor complex in rat brain. In contrast, 3-alpha-hydroxy-5-alpha-pregnan-20-one (3-alpha,5-alpha-P) and 3-alpha-hydroxy-5-beta-pregnan-20-one (3-alpha,5-beta-P) have full efficacy. Moreover, 3-alpha,5-beta-P but not 3-alpha,5-alpha-P recognizes high (nanomolar) and low (micromolar) affinity neuroactive steroid sites in these allosteric modulatory assays. The concentration-response curve for 3-alpha,5-alpha-P modulation of (35S)TBPS binding was shifted rightward in the presence of these pregnanediols and GABA. The maximum shift produced by these pregnanediols never exceeded the concentration-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 recognized 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-alpha-P is only apprx 5-fold. In contrast, the difference between the high (30 nM) and low (7 mu-M) affinity sites discriminated by 3-alpha,5-beta-P is gt 200-fold. Thus, the selective interaction between the high affinity site recognized by 3-alpha,5-beta-P and these pregnanediols can be clearly observed. A saturating concentration of 5-beta-pregnan-3-alpha,20-beta-diol selectively eliminated the high affinity component recognized by 3a,5-beta-P, whereas 5a-pregnan-3-alpha,20-alpha-diol did not completely abolish the high affinity site. 5-alpha-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 (35S)TBPS binding. Collectively, these studies provide evidence that the limited efficacy of certain pregnanediols as allosteric modulators of (35S)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 (35S)TBPS binding to recombinantly expressed receptors suggest that the subunit composition of the GABA-A receptor complex may contribute to pregnanediol selectivity. The relative contributions of neuroactive steroid receptor subtypes and/or different affinity states of the same receptor to the apparent receptor heterogeneity observed remain to be determined.