Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites.
Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites.
复制标题
大鼠脑烟碱型α-金环蛇毒素受体与稳定表达的α-金环蛇毒素结合位点之间的相似性。
DOI：
10.1046/j.1471-4159.1996.67010145.x
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发表时间：
1996
影响因子：
4.7
通讯作者：
Puchacz,E
中科院分区：
文献类型：
--
作者：
Quik,M;Choremis,J;Komourian,J;Lukas,RJ;Puchacz,E
目前的结果表明，α-银环蛇毒素（α-BGT）结合位点的GH4 C1大鼠垂体克隆系细胞的稳定表达。野生型GH4 C1细胞不表达α-BGT结合位点，也不含可检测的烟碱受体α2、α3、α4、α5、α7、β2或β3亚基mRNA。而稳定转染大鼠烟碱受体α7 cDNA的GH4 C11细胞（α7/GH4 C11细胞）大量表达该转基因的mRNA，北方分析表明该信息与预期大小相符。α7/GH4 C1细胞也表达125 I标记的α-BGT的饱和、高亲和力结合位点，其Kd为0.4 nmandBmax为3.2 fmol/106个完整细胞。125 I-α-BGT结合亲和力和药理学特征在大鼠脑或α7/GH4 C1细胞制备的膜中的位点没有显著差异。此外，完整α7/GH4 C1细胞上的125 I-α-BGT结合位点的KD和Ki值与培养的海马神经元的KD和Ki值基本相似。蔗糖密度梯度分析显示α7/GH4 C1细胞表达的α-BGT结合位点大小与天然脑α-BGT受体相似。培养的α7/GH4 C1细胞长期暴露于尼古丁或升高的细胞外钾浓度可诱导α-BGT结合位点数量的变化，与在培养的神经元中观察到的变化相当。总的来说，目前的结果表明，α7/GH4 C1细胞中α-BGT结合位点的性质与脑烟碱α-BGT受体的性质相似。如果本研究中异源表达的α-BGT结合位点仅由α7亚基组成，则结果可能表明大鼠脑α-BGT受体具有相似的同源寡聚体结构。或者，如果α-BGT结合位点以α7与其他一些先前鉴定的或新的亚基的异源寡聚体形式存在，则数据将表明α7亚基在决定α-BGT受体特性方面发挥主要作用。
The present results demonstrate stable expression of α‐bungarotoxin (α‐BGT) binding sites by cells of the GH4C1rat pituitary clonal line. Wild‐type GH4C1cells do not express α‐BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH4C1cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH4C1cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH4C1cells also express saturable, high‐affinity binding sites for125I‐labeled α‐BGT, with aKDof 0.4 nMandBmaxof 3.2 fmol/106intact cells.125I‐α‐BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH4C1cells. Furthermore,KDandKivalues for125I‐α‐BGT binding sites on intact α7/GH4C1cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α‐BGT binding sites expressed in α7/GH4C1cells was similar to that of the native brain α‐BGT receptor. Chronic exposure of α7/GH4C1cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α‐BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α‐BGT binding sites in transfected α7/GH4C1cells resemble those for brain nicotinic α‐BGT receptors. If the heterologously expressed α‐BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α‐BGT receptor has a similar homooligomeric structure. Alternatively, if α‐BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α‐BGT receptor.