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W. Kothmann, S. C. Massey, J. O'Brien; The Extent of Tracer Coupling Between AII Amacrine Cells is Directly Related to Phosphorylation of Ser293 on Connexin36 Gap Junctions. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1029.
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© ARVO (1962-2015); The Authors (2016-present)
Electrical synapses composed of connexin36 (Cx36) gap junctions couple AII amacrine cells (AII ACs) and are a necessary component of the primary rod pathway (Deans et al., 2002). Coupling between AII ACs is regulated by dopamine signaling (Hampson et al., 1992). We previously demonstrated that phosphorylation of Cx36 at Ser293 in AII ACs is modulated via the D1 dopamine pathway: a D1 antagonist increased phosphoryaltion, while a D1 agonist reduced phosphorylation in a PKA-dependent manner (Kothmann et al., ARVO 2008 #1515). We hypothesize that AII AC coupling is regulated by Cx36 phosphorylation, and that D1 receptor/PKA activation in turn activates a phosphatase.
AII ACs were injected with Neurobiotin in isolated rabbit retina superfused with Ames medium (control), D1 agonist, or D1 antagonist. The retina was fixed and labeled with antibodies against Cx36 and phosphoSer293-Cx36, visualized with fluorescent secondary antibodies and streptavidin, and imaged on a confocal microscope. The diffusion coefficient of the Neurobiotin (kAII-AII) was calculated for each sample using a compartmental diffusion model and correlated with Cx36 phosphorylation, measured as the ratio of phosphoSer293 labeling to Cx36 labeling. Also, retina-sclera pieces were incubated with D1 agonist, phosphatase inhibitor, or D1 agonist plus phosphatase inhibitor. These pieces were labeled as above, except that anti-calretinin antibodies were used to label AII ACs.
A direct relationship was found between Cx36 phosphorylation and AII AC tracer coupling. D1 agonist (SKF38393, 10 µM) significantly reduced both Ser293 phosphorylation and kAII-AII, while D1 antagonist (SCH23390, 10 µM) increased both. Incubation of retina-sclera pieces with the PP2A/PP1 inhibitor microcystin-LR (1 nM) significantly increased Ser293 phosphorylation. Microcystin-LR (1 nM) also blocked the D1 agonist-mediated reduction in Cx36 phosphorylation.
The phosphorylation state of Cx36 gap junctions in AII ACs is directly related to the coupling state of the AII network. Increased phosphorylation is associated with increased coupling. This is consistent with our hypothesis that phosphorylation of Cx36 is the mechanism regulating the permeability of these gap junctions. Activation of D1 dopamine receptors appears to lead to increases in phosphatase activity, which in turn leads to reduced phosphorylation of Cx36. We show evidence that PP2A and/or PP1 activation is one component of this pathway.
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