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J. O'Brien, Y.J. Keflemariam, G.S. Burr, M.B. Snuggs; Connexin35 Is Phosphorylated In The Retina In Vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5386.
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© ARVO (1962-2015); The Authors (2016-present)
Changes in the network properties of electrically coupled neurons are an important component of retinal light adaptation. Electrical synapses in many of these neurons are formed by the gap junction protein connexin35 (Cx35), and changes in coupling through Cx35 gap junctions can influence network properties. We have found that Cx35–mediated coupling is regulated by phosphorylation at specific residues. This study was designed to develop biochemical tools to evaluate the functional state of Cx35 gap junctions.
Polyclonal antibodies were developed against phosphorylated peptides corresponding to two phosphorylation sites critical for the regulation of Cx35 function. Phospho–specific antibodies were prepared by affinity purification with the phosphorylated peptide and depletion with non–phosphorylated peptide. Antibodies were analyzed by western blot and immunofluorescence in cryostat sections of ethanol/formaldehyde fixed zebrafish retina.
Antibodies against phosphorylated Ser110 and phosphorylated Ser276 of perch Cx35 specifically recognized in vitro phosphorylated GST–Cx35 intracellular loop and GST–Cx35 C–terminal domain fusion proteins, respectively. The non–phosphorylated fusion proteins were not recognized. In western blots of light–adapted hybrid bass retinal membranes, both antibodies labeled a prominent band of about 33 kDa that co–migrated with a band labeled by a Cx35 monoclonal antibody. In sections of light–adapted zebrafish retina, the phospho–110 antibody showed abundant punctate labeling in the inner and outer plexiform layers and sparse labeling in the inner nuclear layer. This labeling co–localized extensively with Cx35 monoclonal antibody labeling. However, some labeling in the inner and outer plexiform layers and all labeling in the inner nuclear label did not co–localize with Cx35 monoclonal antibody labeling. Sections of retina from dark–adapted, subjective night zebrafish show significantly less phospho–110 labeling with no apparent change in Cx35 labeling.
Cx35 is phosphorylated in vivo at two sites in light–adapted conditions. Antibodies to phospho–Ser110 label phosphorylated Cx35 gap junctions, but also label some non–Cx35 structures. Labeling of all of these structures depends on the state of light adaptation, suggesting that phosphorylation changes dynamically.
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