Drafts Brandon C, Fisher Janet L
Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina School of Medicine, Columbia, SC 29208, USA.
J Pharmacol Exp Ther. 2006 Sep;318(3):1094-101. doi: 10.1124/jpet.106.104844. Epub 2006 May 25.
Barbiturates act on GABA(A) receptors (GABARs) through three distinct mechanisms, resulting in positive allosteric modulation, direct activation, and inhibition. These effects are observed at different concentrations and are differentially affected by some mutations and by the receptor's subunit composition. Mammalian GABARs can be formed from a combination of 16 different subunit subtypes. Although the effect of barbiturates depends largely on the beta subunit, their agonist activity is substantially influenced by the alpha subunit subtype. Pentobarbital is a more effective agonist than GABA only when receptors contain an alpha6 subunit. Results from chimeric alpha1/alpha6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for this characteristic. Within this domain, we examined 15 amino acid residues unique to the alpha6 subtype. Each of these sites was individually mutated in the alpha6 subunit to the corresponding residue of the alpha1 subunit. The effect of the mutation on direct activation by pentobarbital was determined with whole-cell electrophysiological recordings. Our results indicate that only one of these mutations, alpha6(T69K), altered pentobarbital efficacy. This single mutation reduced the response to pentobarbital to a level intermediate to the wild-type alpha1beta1gamma2L and alpha6beta1gamma2L isoforms. The mutation did not affect the sensitivity of the receptor to GABA but did reduce the efficacy of etomidate, another i.v. anesthetic with activity similar to pentobarbital. The reverse mutation in the alpha1 subunit (K70T) did not alter the response to pentobarbital. This is the first identification of a structural difference in GABAR alpha subtypes that regulates direct activation by barbiturates.
巴比妥类药物通过三种不同机制作用于GABA(A)受体（GABARs），产生正变构调节、直接激活和抑制作用。这些效应在不同浓度下观察到，并且受到一些突变和受体亚基组成的不同影响。哺乳动物GABARs可由16种不同亚基亚型组合形成。尽管巴比妥类药物的作用在很大程度上取决于β亚基，但其激动剂活性受到α亚基亚型的显著影响。只有当受体含有α6亚基时，戊巴比妥才是比GABA更有效的激动剂。嵌合α1/α6亚基的结果表明，细胞外N端结构域内的结构差异是造成这一特性的原因。在该结构域内，我们检查了α6亚型特有的15个氨基酸残基。这些位点中的每一个在α6亚基中都被单独突变为α1亚基的相应残基。通过全细胞膜片钳电生理记录确定突变对戊巴比妥直接激活的影响。我们的结果表明，这些突变中只有一个，即α6(T69K)，改变了戊巴比妥的效力。这一单一突变将对戊巴比妥的反应降低到野生型α1β1γ2L和α6β1γ2L亚型之间的水平。该突变不影响受体对GABA的敏感性，但确实降低了依托咪酯的效力，依托咪酯是另一种静脉麻醉药，其活性与戊巴比妥相似。α1亚基中的反向突变（K70T）并未改变对戊巴比妥的反应。这是首次鉴定出GABAR α亚型中调节巴比妥类药物直接激活的结构差异。
