Batuecas A, Cubero A, Barat A, Ramírez G
Centro de Biologia Molecular (CSIC-UAM), Canto Blanco, 28049 Madrid, Spain.
Neurochem Int. 1987;10(4):405-11. doi: 10.1016/0197-0186(87)90066-0.
As part of an ongoing study on the GABA(A) receptor complex in the developing chick optic tectum we describe some properties of the agonist site, as labeled by [(3)H]muscimol, including methodological, kinetic and pharmacological aspects. 16-day embryos and 10-day chicks have been selected as representative age points for the initial characterization of the receptor, prior to more detailed developmental studies. Our data indicate the existence, in both embryos and young birds, of a single class of statistically equivalent, high-affinity, saturable binding sites, with a dissociation constant (K(d)) of 80-90 nM in freeze-thawed/washed membranes, and about 8 nM in membranes additionally extracted with low concentrations of Triton X-100. Maximal densities of binding sites are nearly identical in both membrane preparations, ranging from ?2 to ?3 pmol/mg for the two age points considered. The pharmacological profiles suggest that avian receptors for [(3)H]muscimol are generally similar to the corresponding mammalian sites, behaving as typical bicuculline-sensitive, baclofen-insensitive type A GABA receptor sites. However, bicuculline and its derivatives are less efficient displacers of [(3)H]muscimol in detergent-extracted membrane preparations, being in all cases, as usually, much less effective displacers than GABA agonists. The effect of Triton X-100 on the muscimol site in the GABA(A) receptor, increasing the affinity for the radioligand by a factor of 10, and diminishing the efficiency of antagonists, is considered here in terms of structural changes in the receptor, induced by the action of the detergent on the membrane microenvironment.
作为一项正在进行的关于发育中的雏鸡视顶盖中GABA(A)受体复合物研究的一部分，我们描述了由[(3)H]蝇蕈醇标记的激动剂位点的一些特性，包括方法学、动力学和药理学方面。16日龄胚胎和10日龄雏鸡被选为受体初步表征的代表性年龄点，以便进行更详细的发育研究。我们的数据表明，在胚胎和幼鸟中均存在一类统计学上等效的、高亲和力的、可饱和的结合位点，在冻融/洗涤的膜中解离常数(K(d))为80 - 90 nM，在另外用低浓度Triton X - 100提取的膜中约为8 nM。两种膜制剂中结合位点的最大密度几乎相同，所考虑的两个年龄点的范围为每毫克约2至3 pmol。药理学特征表明，[(3)H]蝇蕈醇的禽类受体通常与相应的哺乳动物位点相似，表现为典型的对荷包牡丹碱敏感、对巴氯芬不敏感的A型GABA受体位点。然而，在去污剂提取的膜制剂中，荷包牡丹碱及其衍生物作为[(3)H]蝇蕈醇的置换剂效率较低，在所有情况下，与GABA激动剂相比，通常都是效果差得多的置换剂。本文从去污剂对膜微环境的作用诱导受体结构变化的角度，考虑了Triton X - 100对GABA(A)受体中蝇蕈醇位点的影响，即对放射性配体的亲和力增加了10倍，并降低了拮抗剂的效率。
