期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 46, 期 9, 页码 3247-3255出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.04-1504
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资金
- NEI NIH HHS [EY 040110, EY 13421] Funding Source: Medline
PURPOSE. Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 ( Cx46) and connexin50 ( Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase C gamma ( PKC gamma) affects the number of channels composed of Cx46, Cx50, or both connexins. METHODS. Whole rat lenses were treated with phorbol- 12- myristate-13- acetate ( TPA) to activate PKC gamma or the inactive analogue 4 alpha- phorbol, 12,13- didecaneote ( PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze- fracture immunolabeling ( FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKC gamma activity, connexin phosphorylation and coimmunoprecipitation. RESULTS. Treatment with TPA activated PKC gamma and uncoupled the lens cortex by similar to 60%. PDD had no effect. Activation of PKC gamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKC gamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin ( Cav)- 1. In the membrane environment, PKC gamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines. CONCLUSIONS. The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.
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