Chimeric GABAA/glycine receptors: expression and barbiturate pharmacology.
Chimeric GABAA/glycine receptors: expression and barbiturate pharmacology.
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嵌合 GABAA/甘氨酸受体：表达和巴比妥药理学。
DOI：
10.1016/s0028-3908(96)00088-3
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发表时间：
1996
影响因子：
4.7
通讯作者：
Harrison,NL
中科院分区：
文献类型：
--
作者：
Koltchine,VV;Ye,Q;Finn,SE;Harrison,NL
gabaa和甘氨酸受体是配体门控离子通道“基因超家族”中的近亲，但具有明显不同的药理学。例如，巴比妥类药物对gabaa受体（GABAA-R）有两种作用：在低微摩尔浓度（2-5 μM）下，麻醉剂巴比妥类药物甲己ital增强了对GABA的亚极大氯电流反应；在较高浓度（20 ~ 50 μM）下，巴比妥酸盐在没有激动剂的情况下直接门控通道。巴比妥酸盐对甘氨酸受体（Gly-R）均无影响。为了研究GABAA-R参与巴比土类药物药理作用的结构部分，我们在编码人GABAA-R α2和β1亚基以及人gly-R α1亚基的cdna中引入了两个独特的限制性位点。第一个位点（“X”）对应于每个亚基中第三个跨膜结构域（M3）的c端末端，并使GABAA-R和Gly-R亚基之间的c端片段（包括大的“细胞质环”和M4片段）能够交换约100个氨基酸。第二个位点（' S ‘）距离每个亚基的n端约30个氨基酸3 ’ -，使GABAA-R和Gly-R亚基之间的小n端片段交换成为可能。构建了几种嵌合受体亚基cdna，并测试了所得到的受体对GABA和甘氨酸的反应能力以及对巴比妥酸盐（MTX）的敏感性。结果表明，无论是大的c端片段还是小的n端片段都与MTX增强或直接激活GABAA-R无关。这些结果证明了GABAA/Gly-R嵌合物的可行性，为进一步研究GABAA- r的巴比妥酸盐药理作用的分子基础奠定了基础。版权所有©1996 Elsevier Science Ltd。
GABAAand glycine receptors are close relatives in the ‘gene superfamily’ of ligand-gated ion channels, but have distinctly different pharmacology. For example, barbiturates have two effects on GABAAreceptors (GABAA-R): at low micromolar concentrations (2–5 μM), the anesthetic barbiturate methohexital potentiates submaximal chloride current responses to GABA; at higher concentrations (20–50 μM), the barbiturate causes direct gating of the channel in the absence of agonist. Neither of these barbiturate effects is seen on the glycine receptor (Gly-R). In order to study the structural parts of the GABAA-R involved in this barbiturate pharmacology, two unique restriction sites were introduced into the cDNAs encoding the α2 and β1 subunits of the human GABAA-R and the α1 subunit of the human gly-R. The first site (‘X’) corresponded to the C-terminal end of the third transmembrane domain (M3) in each subunit and enabled exchange of a C-terminal fragment of ∼ 100 amino acids (which includes the large ‘cytoplasmic loop’ and M4 segment) between GABAA-R and Gly-R subunits. The second site (‘S’) was ∼ 30 amino acids 3′- from the N-terminal end of each subunit and enabled exchange of a small N-terminal fragment between GABAA-R and Gly-R subunits. Several chimeric receptor subunit cDNAs were constructed and the resulting receptors tested for their ability to respond to GABA and glycine and for sensitivity to the barbiturate methohexital (MTX). The results show that neither the large C-terminal fragment nor the smaller N-terminal fragment is associated with the enhancement or direct activation of the GABAA-R by MTX. These results demonstrate the viability of chimeric GABAA/Gly-R and suggest that the method will be suitable for further investigation of the molecular basis of the barbiturate pharmacology of the GABAA-R. Copyright © 1996 Elsevier Science Ltd.