Tyndale R F, Hales T G, Olsen R W, Tobin A J
Department of Pharmacology, School of Medicine, University of California, Los Angeles 90024.
J Neurosci. 1994 Sep;14(9):5417-28. doi: 10.1523/JNEUROSCI.14-09-05417.1994.
We have investigated the GABAA receptor mRNA composition in 13 cell lines, using 13 subunit-specific oligo-primers (alpha 1-6, beta 1-3, gamma 1-3, and delta) and reverse transcriptase PCR amplification. Cell lines (B35, B65, B103, B104, RINm5F, Rat1, PC12, C6, C17, C27, beta TC3, NB41A3, AtT-20), derived from diverse tissue origins, were investigated in order to identify homogeneous cellular sources with distinctive GABAA receptor subunits. Fifteen GABAA receptor subunits have been cloned from mammalian tissue (those listed above plus the retinal subunits rho 1 and rho 2). This multiplicity of GABAA receptor subunits underlies the diverse pharmacology of the GABAA receptor. Attempts to understand the regulation and pharmacology of individual subunits and of the heterooligomeric receptor combinations have been impeded by a lack of pure populations of cells expressing GABAA receptor subunits. Permanent cell lines provide such a resource. Each GABAA receptor subunit mRNA, alpha 1-5, beta 1-3, gamma 1-3, and delta, was detected in at least one cell line. All cell lines examined contained detectable levels of at least one GABAA receptor subunit mRNA. Each cell line contained distinctive combinations of subunit mRNAs. None of the cell lines examined contained detectable amounts of alpha 6 mRNA. These cell lines, which transcribe GABAA receptor subunit mRNAs, provide useful cellular sources for transcriptional and pharmacological studies. Our data also suggest that endogenous GABAA receptor subunit mRNAs may be present in cells that are routinely used for transfection studies, and that this expression might confound interpretation of the studies. In the following companion article, we have looked for functional GABAA receptor Cl- ion channels in these cell lines, using the patch-clamp technique (Hales and Tyndale, 1994).
我们使用13种亚基特异性寡核苷酸引物(α1 - 6、β1 - 3、γ1 - 3和δ)及逆转录酶PCR扩增技术,研究了13种细胞系中的GABAA受体mRNA组成。为了鉴定具有独特GABAA受体亚基的同质细胞来源,我们对源自不同组织的细胞系(B35、B65、B103、B104、RINm5F、Rat1、PC12、C6、C17、C27、βTC3、NB41A3、AtT - 20)进行了研究。已从哺乳动物组织中克隆出15种GABAA受体亚基(上述列出的亚基加上视网膜亚基rho 1和rho 2)。GABAA受体亚基的这种多样性是GABAA受体多种药理学特性的基础。由于缺乏表达GABAA受体亚基的纯细胞群体,对单个亚基以及异源寡聚体受体组合的调控和药理学研究受到了阻碍。永久性细胞系提供了这样一种资源。在至少一种细胞系中检测到了每种GABAA受体亚基mRNA,即α1 - 5、β1 - 3、γ1 - 3和δ。所有检测的细胞系中都含有可检测水平的至少一种GABAA受体亚基mRNA。每个细胞系都含有亚基mRNA的独特组合。所检测的细胞系中均未检测到α6 mRNA。这些转录GABAA受体亚基mRNA的细胞系为转录和药理学研究提供了有用的细胞来源。我们的数据还表明,内源性GABAA受体亚基mRNA可能存在于常用于转染研究的细胞中,并且这种表达可能会混淆研究的解释。在接下来的配套文章中,我们使用膜片钳技术(Hales和Tyndale,1994)在这些细胞系中寻找功能性GABAA受体氯离子通道。