McCauley L D, Liu V, Chen J S, Hawkinson J E, Lan N C, Gee K W
Department of Pharmacology, College of Medicine, University of California, Irvine 92717.
Mol Pharmacol. 1995 Feb;47(2):354-62.
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 approximately 5-fold. In contrast, the difference between the high (30 nM) and low (7 microM) affinity sites discriminated by 3 alpha,5 beta-P is > 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 3 alpha,5 beta-P, whereas 5 alpha-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.(ABSTRACT TRUNCATED AT 400 WORDS)
研究人员观察到某些内源性孕二醇（5α-孕烷-3α,20α-二醇和5β-孕烷-3α,20β-二醇）作为t-[35S]丁基双环磷硫代酸盐（[35S]TBPS）和[3H]氟硝西泮与大鼠脑γ-氨基丁酸（GABA）A受体复合物位点结合的变构调节剂，其功效有限。相比之下，3α-羟基-5α-孕烷-20-酮（3α,5α-P）和3α-羟基-5β-孕烷-20-酮（3α,5β-P）具有完全功效。此外，在这些变构调节试验中，3α,5β-P而非3α,5α-P能识别高（纳摩尔）和低（微摩尔）亲和力的神经活性甾体位点。在这些孕二醇和GABA存在的情况下，3α,5α-P对[35S]TBPS结合的浓度-反应曲线向右移动。这些孕二醇产生的最大移动从未超过在无GABA时单独使用3α,5α-P获得的浓度-反应曲线。此外，在无GABA时，5α-孕烷-3α,20α-二醇和5β-孕烷-3α,20β-二醇对3α,5α-P识别的位点均无任何影响。3α,5α-P识别的两个明显位点的亲和力差异（分别在有和无GABA时为29 nM对152 nM）仅约为5倍。相比之下，3α,5β-P区分的高（30 nM）和低（7 μM）亲和力位点之间的差异>200倍。因此，可以清楚地观察到3α,5β-P识别的高亲和力位点与这些孕二醇之间的选择性相互作用。饱和浓度的5β-孕烷-3α,20β-二醇选择性地消除了3α,5β-P识别的高亲和力成分，而5α-孕烷-3α,20α-二醇并未完全消除高亲和力位点。相对于5β-孕烷-3α,20β-二醇，5α-孕烷-3α,20α-二醇仅识别3α,5β-P区分的部分高亲和力位点，而这两种孕二醇识别介导3α,5α-P对[35S]TBPS结合抑制作用的相似位点群体。总体而言，这些研究提供了证据，表明某些孕二醇作为[35S]TBPS结合变构调节剂的有限功效可能部分归因于对3α,5β-P识别的高亲和力位点的选择性。（摘要截短于400字）
