Due to its unique structure and function, lens metabolism and ionic homeostasis depend absolutely on the lens fibers remaining interconnected to the surface epithelial cells by gap junctional communication pathways. Ionic homeostasis is essential in order to avoid cataract: precipitation of the high concentration of soluble proteins in the lens fiber cytoplasms. The long-term objectives of this proposal are to experimentally demonstrate the function of intercellular communication via gap junctions in lens development and homeostasis. Experiments are outlined which are designed carry out specific blocks of connexin functions in the lens. A Cx43 knockout mouse is now available commercially. These animals die at birth of cardiac defects thus living long enough to permit a study of the developmental and homeostatic sequellae of a knockout of Cx43, which is expressed first in normal lens development, and which persists in the mature lens in both epithelial cells and differentiating fibers. To block the function of other lens connexins, trans-dominant negative constructs which interfere with connexin function will be expressed in the mouse as transgenes driven by the alphaA-crystallin promoter, to target expression to the lens. In the chick, active trans-dominant negative constructs will be introduced into the developing lens by retroviral infection, using the replication- competent RCAS-A avian virus. In a second specific aim, phosphorylation sites on connexins will be mapped by conventional peptide mapping and sequencing. Key phosphorylated serine and threonine residues identified by this mapping will be mutated and epitope tagged. These connexin mutants will be introduced into lens cells in culture by transfection and into whole lenses using the RCAS retrovirus. The ability of the mutated connexins to assemble into channels, to be transported to the plasma membrane and to target to gap junctions will be followed by immunohistochemistry in both the cultures and in developing lenses. Transfection of these mutant connexins into the communication-negative Neuro2A neuroblastoma cell line will permit the study of changes in single channel conductance, voltage gating, and pH sensitivity resulting from the loss of specific phosphorylated residues.
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