Expression and modulation of channel proteins in neuronal gap junctions.

连接蛋白构成缝隙连接的蛋白质亚单位。本研究对参与电紧张性突触的神经元连接蛋白在成年哺乳动物视网膜和大脑中存在缝隙连接网络的区域的表达和定位进行了研究。通过逆转录-聚合酶链式反应(RT-PCR)从Wistar大鼠视网膜总RNA中克隆了神经元连接蛋白候选基因Cx36。为了确定Cx36表达的细胞来源，针对根据Cx36基因序列推导出的321个氨基酸序列的Cx36胞质环预测的合成肽，在兔中制备了抗体。亲和纯化的抗体识别来自视网膜、嗅球、海马体、新皮质、小脑、延髓和脊髓的组织提取物中的一种36 kDa蛋白质。在视网膜和…的内丛状层发现Cx36免疫反应阳性。免疫细胞化学标记显示脑区有更多的D。我们将亚克隆到载体中的大鼠Cx36基因导入COS细胞，发现Cx36基因和蛋白在COS细胞中瞬时表达，这些细胞在细胞间形成了功能性的细胞间通道。视网膜无长突细胞和神经节细胞、海马和新皮质非锥体细胞、嗅球颗粒细胞的胞体和树突呈胞浆免疫反应。RT-PCR分析表明，发育和成人大脑区域，包括新皮质，都表达一定水平的Cx36基因转录。Cx36-构建缝隙连接的细胞间通道可能通过大脑和视网膜中的耦合神经元提供广泛的神经元信号传播。缝隙连接的通透性是由连接蛋白的磷酸化通过蛋白激酶的激活来调节的。我们预测了Cx36蛋白中潜在的磷酸化位点。研究Cx36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。较少

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