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Expression and modulation of channel proteins in neuronal gap junctions.

神经元间隙连接中通道蛋白的表达和调节。

基本信息

批准号：
11680794
负责人：
MIYACHI Ei-ichi
金额：
$ 2.37万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680794/
关键词：
retina brain gap junction connexin family connexin 36 RT-PCR Western blot intercellular communication 細胞間連絡

项目摘要

Connexins constitute the protein subunits of gap junctions. In the present study the expression and localization of neuronal connexins participating in electrotonic synapses were examined in adult mammalian retina and brain regions where gap junction networks of neurons are present. Connexin 36 (Cx36), a candidate for neuronal connexin, was cloned to amplify a cDNA by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA in Wister rat retina. To define cellular origin of Cx36 expression, antibodies were raised in rabbits directed against a synthetic peptide corresponding to the predicted cytoplasmic loop of the Cx36 with the 321 amino acid alignment, deduced from the Cx36 cDNA sequence. The affinity-purified antibodies recognized a 36-kDa protein in tissue extract from the retina, olfactory bulb, hippocampus, neocortex, cerebellum, medulla and spinal cord in Western blots. Cx36-immunoreactivity was found as numerous puncta in the inner plexiform layer of the retina an … More d in the brain regions by immunocytochemical labeling. We identified transient expression of Cx36 mRNA and proteins when the rat Cx36 cDNA subcloned into a vector was introduced into COS cells, and these transfected cells formed functional intercellular channels between the cells. Somata and dendrites from amacrine and ganglion cells in the retina, from nonpyramidal cells in the hippocampus and neocortex, and from granular cells in the olfactory bulb demonstrated cytoplasmic immunoreactivity. Developing and adult brain regions including the neocortex were shown to express certain level of Cx36 mRNA transcript by RT-PCR analysis. Cx36-intercellular channels constructing gap junctions may provide extensive spread of neuronal signals through coupled neurons in the brain as well as in the retina. Permeability of gap junctions is regulated by phosphorylation of connexin proteins via activation of protein kinases. We predicted potential phosphorylated sites in the Cx36 protein. Study of phosphorylation of the Cx36 protein is necessary for regulation of intercellular communication between neurons. Less

连接蛋白构成缝隙连接的蛋白质亚基。在本研究中，参与电紧张性突触的神经元连接蛋白的表达和定位在成年哺乳动物的视网膜和大脑区域的神经元的缝隙连接网络存在的检查。从大鼠视网膜总RNA中克隆了神经元连接蛋白Cx 36的cDNA。为了确定Cx 36表达的细胞起源，在兔中产生针对合成肽的抗体，所述合成肽对应于从Cx 36 cDNA序列推断的具有321个氨基酸比对的Cx 36的预测胞质环。亲和纯化的抗体在Western印迹中识别来自视网膜、嗅球、海马、新皮质、小脑、髓质和脊髓的组织提取物中的36-kDa蛋白。Cx 36免疫反应在视网膜的内丛状层中被发现为许多斑点， ...更多信息 d在脑区的免疫细胞化学标记。我们发现，Cx 36的mRNA和蛋白质的瞬时表达时，大鼠Cx 36的cDNA亚克隆到一个载体被引入到COS细胞，这些转染的细胞形成细胞间的功能通道。体细胞和树突无长突和神经节细胞在视网膜中，从非锥体细胞在海马和新皮质，并从颗粒细胞在嗅球表现出细胞质免疫反应。RT-PCR分析表明，发育和成年大脑区域，包括新皮层表达一定水平的Cx 36 mRNA转录本。构建缝隙连接的Cx 36-细胞间通道可以通过脑中以及视网膜中的耦合神经元提供神经元信号的广泛传播。间隙连接的通透性通过连接蛋白的磷酸化经由蛋白激酶的活化来调节。我们预测了Cx 36蛋白中潜在的磷酸化位点。研究Cx 36蛋白的磷酸化对于调节神经元之间的细胞间通讯是必要的。少