Institut für Genetik der Universität Bonn, Römerstr, Bonn, Germany.
BMC Ophthalmol. 2010 Oct 27;10:27. doi: 10.1186/1471-2415-10-27.
Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.
Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.
Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.
In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.
缝隙连接通道允许各种哺乳动物组织中相邻细胞之间进行直接的代谢和电偶联。每个通道由 12 个蛋白质亚基组成，称为连接蛋白（Cx）。在小鼠视网膜中，Cx43 主要位于星形胶质细胞之间，而 Cx36、Cx45 和 Cx57 基因的表达已在不同的神经元亚型中检测到。然而，在人视网膜中，连接蛋白基因的表达模式在很大程度上是未知的。
通过 Northern blot 杂交、RT-PCR 和免疫荧光分析，至少部分地探讨了以下人类连接蛋白基因 GJD2（hCx36）、GJC1（hCx45）、GJA9（hCx59）和 GJA10（hCx62）在人视网膜中的表达模式。
在这里，我们报告说，在人视网膜 RNA 中发现了与 hCx36 和 hCx45 同源的 Northern blot 杂交信号。两种连接蛋白的免疫荧光信号都可以位于内丛状层（IPL）和外丛状层（OPL）中。通过 Northern blot 杂交在人视网膜中还检测到了第三个连接蛋白基因 GJA10（Cx62）的表达。有趣的是，其基因结构与在小鼠水平细胞中表达的 Gja10（mCx57）相似，在其外显子 2 下游约 25kb 的额外外显子进一步剪接，编码 12 个氨基酸残基，进一步接近 GJA10（Cx62）的完整阅读框。然而，与斑马鱼 Cx55.5 具有高度序列同一性的 Cx59 mRNA 仅通过 RT-PCR 在人视网膜 cDNA 中弱可检测。
与在小鼠中表达的神经元连接蛋白基因 Gjd2（编码 mCx36）、Gjc1（编码 mCx45）和 Gja10（编码 mCx57）不同，在人视网膜中至少可以检测到包括独特的 GJA9（Cx59）和 GJA10（Cx62）在内的一组 4 个连接蛋白基因的转录本同工型。首次免疫荧光分析显示 hCx36 和 hCx45 表达在 IPL 和 OPL 中的染色模式，部分类似于在小鼠中的表达模式，尽管需要更多的死后材料来进一步探索它们的亚层特异性分布。针对 Cx59 和 Cx62 蛋白的适当抗体将在未来的研究中阐明这些蛋白的表达。
