A recent study shows that mice deficient in the lens gap junction protein connexin 46 (Cx46) develop cataracts, indicating the direct role of gap junction-mediated communication in maintaining lens transparency. Gap junctions are clusters of transmembrane channels that connect the cytoplasm of adjacent cells. These channels permit small metabolites, ions and second messengers to pass from cell to cell. Lens fiber cells within the interior of the vertebrate eye lens have nether a blood supply nor organelles. Thus, lens survival and homeostasis are uniquely dependent on intercellular communication via gap junctions with epithelial cells at the lens surface. The chick lens is chosen as an experimental system as it provides many advantages compared with other species. Three connexins are known to participate in the formation of lens gap junction channels: Cx43 in lens epithelial cells, Cx45.6 and Cx56 mainly in lens fibers. Lens fiber proteins, which survive the whole life span of animals, are susceptible to post-translational phosphorylation. Phosphorylation of lens connexins may have functional implications in the regulation of lens gap junction channels. Moreover, evidence from other species has shown that phosphorylation of connexins is related to gap junction channel assembly and gating. The first goal of this proposal is to determine the functional role of connexin phosphorylation in the lens which includes: identifying phosphorylation sites, studying functional roles of connexin phosphorylation, and determining the relevant kinases involved. The second objective involves experiments to determine the signals for the correct targeting by different lens connexins and the formation of heteromeric connexins. The final objective involves the characterization of relationships between lens connexins and MIP and the function of MIP in lens gap junctions. The proposed experiments will be performed in established systems, including intact embryonic chicken lenses, primary lens cultures, connexin-deficient cell lines and Xenopus oocytes, In addition, a novel retroviral assay system, newly developed by the applicant, will be employed in which exogenous connexins will be introduced into the embryonic chick lens in situ in order to define the roles of lens gap junctions in lens physiology and pathology.
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