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T. W. White, L. Li, C. Sellitto; Altered Post-Translational Modification of Lens Connexins in Cx46 Knockin Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3637.
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Connexins normally undergo developmentally regulated phosphorylation and proteolytic truncation of their carboxy-termini, and these posttranslational modifications influence channel activity. When Cx46 channels were expressed on the Cx50 gene locus in knockin mice, they altered permeability in epithelial cells and increased junctional conductance in fibers, functional changes that altered cataract phenotypes. To determine if connexin post-translational processing differed in the knockin mice, we compared the pattern of Cx43 and Cx46 phosphorylation and Cx46 proteolytic cleavage in wildtype and knockin lenses.
Full-length Cx46 was detected in paraffin sections of lenses and on western blots of lens membrane fractions with an antibody raised against the last six amino acids of the carboxy terminus. Phosphorylation of Cx43 and Cx46 was compared by western blotting samples treated with alkaline phosphatise in the presence or absence of inhibitors. Epithelial cell permeability was assayed by intercellular transfer of fluorescent dyes.
Cx46 C-terminal cleavage was delayed in Cx46 knockin lenses. In wildtype mice, Cx46 staining using an antibody to the C-terminal tail was strongest in the differentiating fibers and is lost at the transition to mature fibers. In knockin mice, Cx46 staining was detected earlier in the elongating epithelial cells and persisted, at a reduced level, into the mature fibers. Quantitative western blotting showed a greater accumulation of full-length Cx46 in knockin lenses that would be predicted by an equimolar replacement of Cx50. Cx46 was phosphorylated in epithelial cells in knockin mice and the presence of Cx46 in the epithelia induced a novel shift in Cx43 gel mobility that was eliminated by alkaline phosphatise treatment.
When Cx46 channels were expressed on the Cx50 gene locus cleavage of their carboxy-termini was delayed, and this alteration of post-translational processing correlated with a conductance increase in the mature fibers. Expression of Cx46 in lens epithelial cells also altered patterns of dye permeability, and this was correlated with both the de novo phosphorylation of Cx46 in these cells and a novel phosphorylation of Cx43.
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