EFFECTS OF GABA AND VARIOUS ALLOSTERIC LIGANDS ON TBPS BINDING TO CLONED RAT GABA(A) RECEPTOR SUBTYPES
EFFECTS OF GABA AND VARIOUS ALLOSTERIC LIGANDS ON TBPS BINDING TO CLONED RAT GABA(A) RECEPTOR SUBTYPES
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DOI：
10.1111/j.1476-5381.1994.tb13185.x
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发表时间：
1994-08-01
影响因子：
7.3
通讯作者：
THOMSEN, DR
中科院分区：
文献类型：
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作者：
IM, WB;PREGENZER, JF;THOMSEN, DR
1[S-35]t-丁基二环硫代磷酸酯 (TBPS) 是 GABA(A) 受体印防己毒素位点的高亲和力配体。在这里，我们检查了在 GABA 和几种变构配体（地西泮、甲基 6,7-二甲氧基-4-甲基-β-咔啉-3-羧酸酯 (DMCM)、3 α,21-二羟基-5）存在下，TBPS 与由 α 1、α 3 或 α 6 与 β 2 或 β 2 和 γ 2 亚基组合组成的克隆受体的结合。 α-pregnan-20-one (5 α-THDOC)、戊巴比妥和锌)。通过感染重组杆状病毒，克隆的受体在 SF-9 昆虫细胞中瞬时表达。2 在 αβ 亚型中，纳摩尔浓度的 GABA 增强 TBPS 结合，但微摩尔浓度抑制结合。在各个亚型中，增强或抑制 TBPS 结合的半最大 GABA 浓度分别与高亲和力和低亲和力 GABA 结合位点相关。结合的最大增强也根据α亚型而变化（α3β2>>α1β2>α6β2）。在αβγ亚型中，TBPS结合不受纳摩尔浓度的GABA影响，但被微摩尔浓度的GABA抑制。因此，添加γ2似乎消除了高亲和力GABA位点和TBPS位点之间的构象耦合，并且还改变了低亲和力GABA位点；特别是，抑制 TBPS 结合的半最大 GABA 浓度从 >100 (alpha 6 beta 2) 变为 1 mu M (alpha 6 beta 2 gamma 2)。3变构配体也改变了 TBPS 与敏感 GABA(A) 受体亚型的结合。例如，地西泮仅在α1β2γ2和α3β2γ2亚型中，而5α-THDOC在所有亚型中在没有GABA的情况下增强TBPS结合，并增强GABA的抑制作用。戊巴比妥在我们检查的所有亚型中仅表现出后一种效应。4 DMCM 和 Zn，分别是 αβγ 和 αβ 亚型中 GABA 诱导的 Cl 电流的抑制剂，与激动剂产生相反的作用，在没有 GABA 的情况下减少 TBPS 结合，并减弱（或消除在 Zn 的情况下）GABA 对 TBPS 结合的抑制作用。 5 这些结果表明，GABA 结合位点及其构象耦合与TBPS 位点受到 α 同工型（特别是与 α 1 或 α 3 相比，α 6）和涉及 γ 2 亚基的四元相互作用的不同影响。此外，变构配体对TBPS结合的变化不仅包括对TBPS位点的直接（变构）影响，还包括通过GABA位点的间接影响，并且与先前研究中已知的亚型选择性和功能性一致。
1[S-35]t-butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA(A) receptors. Here we examined TBPS binding to the cloned receptors made of alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses.2 In alpha beta subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the alpha isoform (alpha 3 beta 2>>alpha 1 beta 2>alpha 6 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from >100 (alpha 6 beta 2) to 1 mu M (alpha 6 beta 2 gamma 2).3 Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes. For instance, diazepam only in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes, and 5 alpha-THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined.4 DMCM and Zn, inhibitors of GABA-induced Cl currents in alpha beta gamma and alpha beta subtypes, respectively, produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating (or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding.5 These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the alpha isoform (particularly alpha 6 as compared to alpha 1 or alpha 3) and by quaternary interactions involving the gamma 2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.