One of the most fascinating and clinically important questions in Eye Research is how the lens can maintain transparency and homeostasis in the absence of blood supply and with a limited metabolic capacity. Current models propose that the lens establishes a primitive """"""""circulatory system"""""""" that move ions, water and small molecules throughout the lens. Critical to understanding the mechanisms responsible for this system is the identification and characterization of the channels and transporters comprising the pathways followed by the small molecules, ions and water through the lens. This proposal is directed at unveiling the structure and mechanisms of regulation of the extensive cell-to-cell pathway that communicates directly the cytoplasm of fibers in the lens surface and interior. Such an extensive pathway carries the implicit danger that damage of a single fiber injures the entire lens. To avoid this danger, the pathway is tightly regulated at the cellular and molecular levels. Because of the unique morphology and limited metabolism of the fibers, it is unlikely that regulation involves the opening/closing of cell-to-cell channels or the insertion/retrieval of hemi-channels into the plasma membrane. Consequently, we will test an alternative regulatory mechanism involving protein kinase C phosphorylation, disassembly of channels into hemi-channels and re-distribution into lipid rafts. The principal advantages of this mechanism are the clustering of proteins in the cytoplasmic side of the plasma membrane, amplification of the signaling cascade as well as the fact that lipid rafts formation is dynamic and reversible. Specifically, we propose to identify and quantify connexin43 (Cx43), connexin46 (Cx46), connexin50 (Cx50), caveolin-1 and 2 and the protein kinase C gamma isoform in gap junctions, lipid rafts and the plasma membrane. We will use lenses of rats where phosphorylation has been stimulated with phorbol esters and mice where the PKC has been genetically ablated. We will combine biochemical and freeze-fracture-immuno-labeling (FRIL), a method that identifies proteins in their native environment, with high spatial resolution (approximately 3 nm) and in a quantitative manner. We also propose to study the distribution and interaction of connexin and aquaporin channels in the native fiber environment and in the Xenopus oocyte expression system.
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