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H. Li, W. W. Kothmann, O. Shiraz, J. O'Brien; Light Adaptation Alters the Phosphorylation State of Photoreceptor Gap Junctions in Zebrafish (Danio rerio) Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3053.
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© ARVO (1962-2015); The Authors (2016-present)
Connexin 35 (Cx35) forms gap junctions among cone photoreceptors, and lighting conditions change the degree of coupling between photoreceptors in some species. Two regulatory phosphorylation sites in Cx35, Ser110 and Ser276, play critical roles in regulation of gap junction coupling. The current study targeted the Cx35 gap junctions in OPL, and asked if different lighting conditions change phosphorylation level in Ser110 and Ser276.
Adult zebrafish were maintained on a 12:12-hr light:dark cycle. Dark adaptation studies were conducted in darkness or under infrared light and completed 2 hr prior to light onset, while light adaptation studies were conducted under photopic illumination and finished by 2 hr after light onset. Excised eyecups were superfused with oxygenated Ames’ medium for 15-20 min, followed by fixation and immunohistochemistry. Phosphorylation at Ser110 and Ser276 on Cx35 was evaluated by confocal immunofluorescence microscopy using antibodies against Cx35-phosphoSer110, Cx35-phosphoSer276 and Cx35. Cone gap junction populations were identified by labeling with Zpr1 or by transgenic expression of GFP from the cone arrestin promoter. Staining and data acquisition conditions were fixed and phospho-antibody signals were normalized to total Cx35 labeling.
Cx35 gap junctions in the OPL displayed variable levels of phosphorylation at both sites under both dark- and light-adapted conditions. At the Ser276 site, light adaptation caused a net reduction in normalized phosphorylation level (mean=0.31±0.08, n=8 fish) compared to dark-adapted eyes (mean=0.79±0.07, n=7 fish; t-value=4.2, p<0.001). Changes occurred at the Ser110 site in the same manner (dark-adapted mean=1.02±0.15, n=6; light-adapted mean=0.50±0.06, n=7; t-value=3.47, p<0.01).
The phosphorylation state of identified regulatory residues of Cx35 in the OPL is regulated by light adaptation. This change may reflect changes in gap junction coupling.
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