Expression and modulation of channel proteins in neuronal gap junctions.

连接蛋白构成间隙连接的蛋白质亚基。在本研究中，研究了参与电紧张突触的神经元连接蛋白在成年哺乳动物视网膜和存在神经元间隙连接网络的大脑区域的表达和定位。通过逆转录聚合酶链反应（RT-PCR），从Wister大鼠视网膜总RNA中克隆出连接蛋白36 (Connexin 36, Cx36) cDNA。为了确定Cx36表达的细胞起源，我们在家兔中培养了抗体，抗体指向与Cx36细胞质环对应的合成肽，该肽与Cx36 cDNA序列推断的321个氨基酸比对。亲和纯化的抗体在Western blots中识别视网膜、嗅球、海马、新皮层、小脑、髓质和脊髓组织提取物中的36kda蛋白。通过免疫细胞化学标记，发现cx36 -免疫反应性表现为在视网膜内丛状层有大量的小点，在脑区有更多的小点。将大鼠Cx36 cDNA亚克隆载体导入COS细胞后，观察到Cx36 mRNA和蛋白的瞬时表达，转染后的细胞在细胞间形成功能性的细胞间通道。来自视网膜无突细胞和神经节细胞、海马和新皮层非锥体细胞以及嗅球颗粒细胞的体细胞和树突表现出细胞质免疫反应性。通过RT-PCR分析发现，包括新皮层在内的发育和成人大脑区域表达一定水平的Cx36 mRNA转录物。构建间隙连接的cx36 -细胞间通道可能通过大脑和视网膜中的耦合神经元提供神经元信号的广泛传播。间隙连接的通透性是通过蛋白激酶激活连接蛋白磷酸化来调节的。我们预测了Cx36蛋白的潜在磷酸化位点。研究Cx36蛋白的磷酸化对神经元间细胞间通讯的调节是必要的。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