Protein Kinase A-mediated Phosphorylation of Connexin36 in Mouse Retina Results in Decreased Gap Junctional Communication between AII Amacrine Cells

Sep 8, 2006The Journal of biological chemistry

Protein kinase A reduces communication between specific retinal cells in mice by modifying connexin36

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Abstract

Increased phosphorylation of connexin36 (Cx36) is linked to reduced gap junction permeability in mouse retina.

Cx36, a gap junction protein in AII amacrine cells, is phosphorylated by protein kinase A (PKA).
Phosphorylation occurs at multiple sites, particularly at Ser-110 and Ser-293.
In vitro studies showed that activation of PKA raises Cx36 phosphorylation levels.
In cultured mouse retina, phosphorylation of Cx36 is associated with a decrease in tracer coupling between AII amacrine cells.
These findings suggest that light-induced dopamine signaling may regulate visual adaptation through modulation of Cx36 activity.

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Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.

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Protein Kinase A-mediated Phosphorylation of Connexin36 in Mouse Retina Results in Decreased Gap Junctional Communication between AII Amacrine Cells

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