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W. W. Kothmann, T. Yasumura, J. E. Rash, J. O'Brien; Cx35 Gap Junctions Exist in a Variety of Phosphorylation States in the Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4924.
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© ARVO (1962-2015); The Authors (2016-present)
Coupling through Cx35/36 gap junctions is regulated by phosphorylation at a few key sites, suggesting that the phosphorylation state of a gap junction may report its coupled state. To determine whether gap junction phosphorylation varies in retina we examined the phosphorylation state of Cx35 in fish retina under different lighting conditions.
We performed western blot, confocal immunofluorescence, and freeze-fracture EM immunolabeling (FRIL) analyses using phospho-specific antibodies against Cx35-phosphoSer110 and Cx35-phosphoSer276. Hybrid striped bass and zebrafish retinas were collected either dark-adapted two hours before light onset or light-adapted two hours after light onset.
Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites; labeling was eliminated by alkaline phosphatase digestion. FRIL experiments showed that both phospho-specific antibodies labeled gap junctions that were also labeled by monoclonal anti-Cx35 antibodies. Confocal immunofluorescence analysis showed gap junctions identified with the monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions identified by their large size (up to 8 µm x 3 µm) and location in two bands in the IPL showed a prominent shift from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites followed this pattern. Under both lighting conditions other gap junctions varied from non-phosphorylated to heavily phosphorylated. In daytime, light-adapted zebrafish IPL 17% of Cx35 gap junctions showed no phosphorylation at Ser110 and 26% showed no phosphorylation at Ser276.
Cx35 gap junctions in the inner plexiform layer exist in different phosphorylation states that are at least partially related to the population of gap junctions to which they belong. This state can change with lighting conditions. We predict that changes in the phosphorylation state of Cx35 gap junctions reflect changes in coupling strength, and are likely controlled at the level of individual cell types or possibly individual gap junctions.
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