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W.W. Kothmann, Y.J. Keflemariam, M.B. Snuggs, J. O'Brien; Phosphorylation of Connexin35 Driven by Light and PKA in Zebrafish Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1510.
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© ARVO (1962-2015); The Authors (2016-present)
Connexin35 (Cx35) is responsible for the formation of gap junctions in many neurons in the retina and CNS, and it has been shown to be regulated by PKA phosphorylation. In this study we examined two questions: 1) Is the phosphorylation state of Cx35 regulated by light adaptation? 2) Which signaling pathways affect Cx35 phosphorylation?
Dark–adapted, subjective night zebrafish (Danio rerio) were exposed to white light for 10 minutes. The eyes were then rapidly excised, fixed in ethanol/paraformaldehyde, and cryostat sectioned. Tissue sections were immunostained with a highly specific monoclonal antibody against Cx35 and a polyclonal antibody against Ser110–phosphorylated Cx35 that we have developed. In another experiment, excised eyecups from light–adapted zebrafish were superfused for 15 minutes with drugs affecting cAMP signaling pathways, and then fixed and immunostained as above. Immunofluorescence data were collected for each experiment.
Dark–adapted zebrafish retinas showed sparse Cx35 phosphoSer110–like immunoreactivity (110P–IR) in the inner plexiform layer and variable 110P–IR in the outer plexiform layer. The labeling was punctate and much of it co–localized with specific Cx35 labeling by the monoclonal antibody. 10 minutes of light exposure resulted in a large increase in 110P–IR in the IPL and consistent heavy labeling in the OPL. In the IPL, the number of 110P–IR plaques increased with no change in the distribution of labeling intensities. In light–adapted excised eyecups, superfusion with 20µM Rp–8–cpt–cAMPS, a PKA inhibitor, markedly reduced 110P–IR throughout the retina, compared to controls superfused with normal Ringer.
Stimulation with white light triggers rapid phosphorylation events in vivo in zebrafish retina, including phosphorylation of Cx35 gap junctions. Inhibition of PKA activity perturbs this light–driven phosphorylation, allowing for rapid dephosphorylation. This suggests that retinal light adaptation involves widespread phosphorylation of Cx35, which is known to reduce coupling through this gap junction. PKA activity plays a major role in this regulation.
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