Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles
Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles
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DOI：
10.1523/jneurosci.16-07-02179.1996
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发表时间：
1996-04
期刊：
影响因子：
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通讯作者：
ML Gilmor;NR Nash;A. Roghani;R. Edwards;H. Yi;SM Hersch;A. Levey
中科院分区：
文献类型：
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作者：
ML Gilmor;NR Nash;A. Roghani;R. Edwards;H. Yi;SM Hersch;A. Levey
最近发现了一个胆碱能基因座，它由哺乳动物特有的含有胆碱乙酰转移酶(ChAT)基因的基因组排列和一个可能的囊泡型乙酰胆碱转运体(Vacht)组成。虽然ChAT和Vacht蛋白的转录本已经定位于胆碱能神经元，但对编码的Vacht蛋白知之甚少。在这里，我们描述了用Vacht C末端/谷胱甘肽-S转移酶融合蛋白制备的高特异性兔多克隆抗体的制备，以及天然Vacht蛋白的免疫学特性。这些抗体通过Western blotting特异性识别在HeLa细胞中表达的全长重组Vacht，在55 kDa处有显著的免疫反应带。在大鼠脑匀浆中，在已知的胆碱能神经支配区域，包括纹状体、皮质、海马体和杏仁核，有一条约70 kDa的Vacht免疫反应带。光镜下免疫细胞化学显示胆碱能细胞组有反应产物，非胆碱能区未见反应产物。更重要的是，免疫反应也集中在许多已知接受显著胆碱能神经支配的区域的轴突纤维中，如大脑皮层、海马体、杏仁核、纹状体、几个丘脑核团和脑干区。使用免疫过氧化物酶的电子显微镜显示，Vacht定位于轴突终末，并使用更精确的免疫金技术，定位于突触小泡。在Vacht阳性的包膜中，免疫金颗粒定位于高尔基复合体的细胞质表面。这些发现证实了Vacht蛋白在胆碱能神经元中唯一表达，集中在突触小泡中，至少在C末端表达，正如模型预测的那样，具有拓扑定向。
A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C- terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT- immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.