Kothmann W Wade, Li Xiaofan, Burr Gary S, O'Brien John
Department of Ophthalmology and Visual Science, University of Texas-Houston Medical School, Houston, Texas 77030, USA.
Vis Neurosci. 2007 May-Jun;24(3):363-75. doi: 10.1017/S095252380707037X. Epub 2007 Jul 20.
Connexin 35/36 is the most widespread neuronal gap junction protein in the retina and central nervous system. Electrical and/or tracer coupling in a number of neuronal circuits that express this connexin are regulated by light adaptation. In many cases, the regulation of coupling depends on signaling pathways that activate protein kinases such as PKA, and Cx35 has been shown to be regulated by PKA phosphorylation in cell culture systems. To examine whether phosphorylation might regulate Cx35/36 in the retina we developed phospho-specific polyclonal antibodies against the two regulatory phosphorylation sites of Cx35 and examined the phosphorylation state of this connexin in the retina. Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites, and this labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membrane preparations. Quantitative confocal immunofluorescence analysis showed gap junctions identified with a monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions that could be identified by their large size (up to 32 microm2) and location in the IPL showed a prominent shift in phosphorylation state from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites showed significant changes in this manner. Under both lighting conditions, other gap junctions varied from non-phosphorylated to heavily phosphorylated. We predict that changes in the phosphorylation states of these sites correlate with changes in the degree of coupling through Cx35/36 gap junctions. This leads to the conclusion that connexin phosphorylation mediates changes in coupling in some retinal networks. However, these changes are not global and likely occur in a cell type-specific or possibly a gap junction-specific manner.
连接蛋白35/36是视网膜和中枢神经系统中分布最广泛的神经元间隙连接蛋白。许多表达这种连接蛋白的神经元回路中的电耦合和/或示踪剂耦合受光适应调节。在许多情况下，耦合的调节取决于激活蛋白激酶（如蛋白激酶A）的信号通路，并且在细胞培养系统中已表明连接蛋白35受蛋白激酶A磷酸化的调节。为了研究磷酸化是否可能在视网膜中调节连接蛋白35/36，我们针对连接蛋白35的两个调节性磷酸化位点开发了磷酸化特异性多克隆抗体，并检测了该连接蛋白在视网膜中的磷酸化状态。用杂交条纹鲈视网膜膜制剂进行的蛋白质印迹分析表明，连接蛋白35在丝氨酸110和丝氨酸276位点均被磷酸化，并且这种标记通过碱性磷酸酶消化而消除。小鼠和兔连接蛋白36的同源位点在视网膜膜制剂中也被磷酸化。定量共聚焦免疫荧光分析表明，用单克隆抗连接蛋白35抗体鉴定的间隙连接在丝氨酸110和丝氨酸276位点均具有可变水平的磷酸化。可以通过其大尺寸（高达32平方微米）和在内网状层中的位置鉴定的异常间隙连接显示出磷酸化状态的显著变化，从夜间暗适应视网膜中的高度磷酸化转变为白天光适应视网膜中的弱磷酸化。丝氨酸110和丝氨酸276位点均以这种方式显示出显著变化。在两种光照条件下，其他间隙连接从非磷酸化到高度磷酸化不等。我们预测这些位点的磷酸化状态变化与通过连接蛋白35/36间隙连接的耦合程度变化相关。这得出结论，连接蛋白磷酸化介导了一些视网膜网络中耦合的变化。然而，这些变化并非全局性的，可能以细胞类型特异性或可能是间隙连接特异性的方式发生。
