Thomet U, Baur R, Scholze P, Sieghart W, Sigel E
Department of Pharmacology, University of Bern, Switzerland.
Br J Pharmacol. 1999 Jul;127(5):1231-9. doi: 10.1038/sj.bjp.0702639.
In electrophysiological measurements the beta-carboline ethyl 6-benzyloxy-beta-carboline-3-carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant alpha1beta2gamma2 GABA(A) receptors and binds with high affinity (IC50-1.5 nM) to the [3H]-flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site. At the dual subunit combination alpha1beta2, which lacks the gamma subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination alpha1beta1, ZK 91085 potentiation is strongly reduced as compared to alpha1beta2. In binding studies, ZK 91085 was able to decrease [35S]-TBPS binding in alpha1beta2gamma2 and alpha1beta2 but not in alpha1beta1. This selectivity of ZK 91085 for receptors containing the beta2 isoform over those containing the beta1 isoform is reminiscent of the action of loreclezole. To identify amino acid residues important for the second type of modulation, we functionally compared wild type alpha1beta2 and mutant receptors for stimulation by ZK 91085. The mutation beta2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation beta2Y62L increased stimulation by ZK 91085 3-4 fold, locating an influencing entity of the second type of action of ZK 91085 at an alpha/beta subunit interface. Structural intermediates of ZK 91085 and the beta-carboline abecarnil, the latter of which only slightly potentiated GABA currents in alpha1/beta2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABA(A) receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical beta-carbolines, that confer negative allosteric modulation through the benzodiazepine site.
在电生理测量中，β-咔啉乙酯6-苄氧基-β-咔啉-3-羧酸酯（ZK 91085）对大鼠重组α1β2γ2 GABA(A)受体起正性变构调节剂的作用，并以高亲和力（IC50 - 1.5 nM）与[3H]-氟硝西泮位点结合。氟马西尼能够部分抵消电流调节作用。这些观察结果表明ZK 91085在苯二氮䓬结合位点起作用。在缺乏苯二氮䓬调节所需γ亚基的α1β2双亚基组合中，我们仍然观察到GABA电流增强。因此，ZK 91085通过通道上的另一个位点起作用。在α1β1亚基组合中，与α1β2相比，ZK 91085的增强作用显著降低。在结合研究中，ZK 91085能够降低α1β2γ2和α1β2中[35S]-TBPS的结合，但不能降低α1β1中的结合。ZK 91085对含有β2亚型的受体比对含有β1亚型的受体具有这种选择性，这让人联想到氯雷唑的作用。为了确定对第二种调节类型重要的氨基酸残基，我们在功能上比较了野生型α1β2和突变受体对ZK 91085刺激的反应。消除氯雷唑作用的β2N265S突变也消除了ZK 91085的刺激作用。β2Y62L突变使ZK 91085的刺激作用增加了3 - 4倍，将ZK 91085第二种作用类型的影响实体定位在α/β亚基界面。分析了ZK 91085和β-咔啉阿贝卡尼的结构中间体，后者在α1/β2中仅轻微增强GABA电流，以确定调节的结构要求。因此，与通过苯二氮䓬位点产生负性变构调节的经典β-咔啉不同，ZK 91085通过两个作用位点对GABA(A)受体进行变构刺激：苯二氮䓬位点和氯雷唑位点。
