Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors
Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors
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DOI：
10.1073/pnas.0509903103
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发表时间：
2006-05-30
影响因子：
11.1
通讯作者：
Olsen, Richard W.
中科院分区：
文献类型：
--
作者：
Hanchar, H. Jacob;Chutsrinopkun, Panida;Olsen, Richard W.
尽管 GABA(A) 受体长期以来一直被认为参与介导乙醇 (EtOH) 的作用，但最近才发现含有“非突触”δ 亚基的受体对 EtOH 具有独特的敏感性。在这里，我们发现含有 δ 亚基的受体以高亲和力 (Kd < 10 nM) 结合咪唑并苯二氮卓类 (BZ) 氟马西尼和 Ro15-4513，这与人们普遍认为这些受体对 BZ 不敏感的观点相反。在免疫纯化的天然小脑和重组 6 亚基受体中，酒精拮抗剂 [H-3]Ro15-4513 的结合受到低浓度 EtOH（K-i 约为 8 mM）的抑制。此外，Ro15-45113 的结合被 BZ 位点配体抑制，这些配体已被证明可以逆转 Ro15-45113 的行为酒精拮抗作用（即氟马西尼、β-咔啉甲酸乙酯 (β-CCE) 和 N-甲基-β-咔啉-3-甲酰胺 (FG7142)，但不包括任何经典的 BZ 激动剂，如地西泮）。旨在区分竞争机制和变构机制的实验表明，EtOH 和 Ro15-4513 占据相互排斥的结合位点。事实上，只有 Ro15-4513（而不是氟马西尼）可以抑制 EtOH 效应，并且 Ro15-4513 与氟马西尼的区别仅在于分子中的一个基团（BZ 环 V 位上的叠氮基），这表明 Ro15-45113 中的这个叠氮基可能是与酒精结合位点重叠的区域。我们的研究结果与之前 Ro15-4513 是行为酒精拮抗剂的观察相结合，表明 EtOH 在相关生理浓度下的许多行为效应是由 EtOH/Ro15-4513 敏感的 GABA(A) 受体介导的。
Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (Kd < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant 6 subunit-containing receptors, binding of the alcohol antagonist [H-3]Ro15-4513 is inhibited by low concentrations of EtOH (K-i approximate to 8 mM). Also, Ro15-45113 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-45113 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the V position of the BZ ring) suggest that this azido group in Ro15-45113 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors.