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H. Li, J. O'Brien; Regulation of Photoreceptor Coupling via Connexin35 Phosphorylation in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5173.
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© ARVO (1962-2015); The Authors (2016-present)
Connexin 35 (Cx35) gap junctions are extensively expressed on photoreceptors and can be regulated by light exposure and neurotransmitter signaling pathways. Two phosphorylation sites on Cx35, Ser110 and Ser276, regulate gap junction coupling. We studied the Cx35 gap junctions in the outer plexiform layer (OPL) and examined the physiological and pharmacological conditions that change phosphorylation state of Cx35 and coupling of cone photoreceptors.
Adult zebrafish were maintained on a 12hr light:12hr dark cycle. Night studies were conducted in darkness and completed 2hr prior to light onset; daytime studies were performed under photopic illumination and finished by 2hr after light onset. Isolated eyecups were superfused with Ames’ medium alone or supplemented with 20µM protein kinase A (PKA) activator Sp-8-cpt-cAMPS or 20µM PKA inhibitor Rp-8-cpt-cAMPS for 30 min, followed by fixation. Phosphorylation at Ser110 and Ser276 was evaluated by immunostaining with antibodies against Cx35-phosphoSer110, Cx35-phosphoSer276 and Cx35 followed by confocal microscopy and quantitative image analysis. Photoreceptor coupling was examined by cut loading of Neurobiotin in flat-mount retina with 21min loading and diffusion time. Neurobiotin-loaded cells were visualized with streptavidin-Cy3 and cone-to-cone diffusion coefficients were estimated using a compartmental diffusion model.
Cx35 gap junctions in the OPL of nighttime retina in darkness were extensively phosphorylated at both Ser110 and Ser276 and cones were well coupled. In contrast, Cx35 was significantly less phosphorylated at both sites in daytime retina and cones were very poorly coupled. Pharmacological stimulation of PKA activity in the day reversed the daytime effects by significantly enhancing Cx35 phosphorylation and cone-cone coupling. Inhibition of PKA activity at night mimicked the daytime effects, significantly reducing Cx35 phosphorylation.
Photoreceptor gap junctions undergo changes in phosphorylation state and coupling competency associated with night:day cycles in zebrafish retina. PKA activity is positively correlated with Cx35 phosphorylation and cone-cone coupling, consistent with a model in which PKA phosphorylates Cx35 directly to support coupling. This behavior is opposite that of AII amacrine cell Cx36 gap junctions, which are closed by PKA activity through the action of an intervening phosphatase (see Kothmann et al., this meeting).
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