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G.A. Zampighi, A.M. Planells, D. Lin, D. Takemoto; Regulation of Lens Gap Junction Communication by Phosphorylation, CX50 Channel Disassembly and Lipid Raft Formation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1131.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the mechanism by which phosphorylation regulates lens fiber gap junction channels composed of equal amounts of connexin46 (Cx46) and connexin50 (Cx50) in the lens cortex. Methods: Whole rat lenses were incubated with the phorbol–12–myristate–13–acetate (TPA) or the inactive 4a–phorbol, 12,13–didecaneote (PDD). The gap junctions were studied with a number of functional, structural and chemical techniques, including dye coupling, Cx46 and Cx50 immuno–fluorescence, freeze–fracture–immuno–labeling (FRIL), kinase activity measurement, immuno–precipitation and sucrose density centrifugation. Results: Treatment with TPA activated the C protein kinases (PKC), especially the PKCγ, and partially uncoupled (∼60%) cortical fiber cells allowing the passage of Lucifer Yellow. Treatment with the inactive phorbol ester PDD had no effect. FRIL showed that the activation of PKCγ decreased the number of Cx50 channels assembled in the gap junction plaques while increasing the number of Cx50 hemi–channels in the fiber plasma membrane. The unlabeled Cx46 channels were redistributed into lipid rafts that appeared as circular voids with negative curvature and an exceedingly small number of integral proteins. PKCγ, the fiber connexins and the protein marker caveolin–1 (Cav–1) were re–located into lipid raft fractions. Cx50 was phosphorylated at serines and threonines; Cx46 was phosphorylated only at threonines. Conclusions: Our studies support the hypothesis that the disassembly of the Cx50 gap junction channels and the redistribution of Cx46 channels into lipid rafts are key mechanisms regulating the cell–to–cell pathway allowing communication between nuclear fibers and metabolically active fibers in the lens surface.
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