Maitra R, Reynolds J N
Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, Canada.
Can J Physiol Pharmacol. 1998 Sep;76(9):909-20. doi: 10.1139/cjpp-76-9-909.
Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.
神经活性甾体是中枢神经系统中γ-氨基丁酸A型（GABA(A)）受体功能的强效、选择性变构调节剂，可能作为内源性抗焦虑和镇痛剂。为了研究GABA(A)受体亚基亚型对神经活性甾体调节受体功能的影响，我们在非洲爪蟾卵母细胞中表达了人重组GABA(A)受体。使用双电极电压钳记录技术测量GABA激活的膜电流，以及内源性神经甾体5α-孕烷-3α-醇-20-酮（别孕烷醇酮）和合成甾体麻醉剂5α-孕烷-3α-醇-11,20-二酮（阿法沙龙）的调节作用。别孕烷醇酮对α1β1γ2L和α1β2γ2L受体亚型增强GABA激活的膜电流具有相似的作用。相比之下，阿法沙龙作为α1β1γ2L受体亚型的正变构调节剂更有效。在没有γ2L亚基亚型的情况下，别孕烷醇酮具有更高的效力，但其效能降低。与α1β2受体亚型相比，别孕烷醇酮对α1β1受体亚型更有效。在没有γ2L亚基亚型的情况下，阿法沙龙增强GABA反应的效力没有改变，尽管其效能大大增强。在没有γ2L亚基的情况下，别孕烷醇酮和阿法沙龙均使GABA浓度-反应关系产生非平行向左移位，降低了GABA的半数有效浓度（EC50）并增加了最大反应。当存在γ2L亚基亚型时，只有阿法沙龙增加了最大GABA反应。3β-孕烷异构体表孕烷醇酮和异孕烷醇酮均抑制别孕烷醇酮和阿法沙龙增强GABA(A)受体功能的能力。然而，3β-孕烷甾体异构体产生的阻断程度取决于所研究的受体亚型类型和所测试的神经活性甾体。在所有测试的受体亚型中，异孕烷醇酮（别孕烷醇酮的3β-异构体）作为别孕烷醇酮引起的增强作用的阻断剂比阿法沙龙更有效。与α1β1γ2L受体亚型相比，表孕烷醇酮作为α1β2γ2L受体亚型的阻断剂具有更高的效能，并且与阿法沙龙相比，对别孕烷醇酮引起的增强作用也产生了更大程度的阻断。我们的结果支持以下假设：含有不同亚基亚型的不同GABA(A)受体亚型的异聚体组装导致GABA(A)受体上有多个甾体识别位点，这反过来又在作用于GABA(A)受体的神经活性甾体之间产生明显不同的调节相互作用。α和γ亚基亚型可能对别孕烷醇酮调节GABA(A)受体功能影响最大，而β和γ亚基亚型似乎对阿法沙龙的调节作用最为重要。
