The fiber cells in the vertebrate lens are connected by unusually extensive intercellular junctions. It has been suggested that these junctions contain cell-to-cell channels, based on demonstrations of the intercellular movement of small molecules within the lens. The functions of the channels in the lens are not understood, although it has been proposed that they provide critical pathways for the movement of metabolites and regulatory molecules. To gain insights into the functional aspects, the regulation of the channels will be examined here. Ultimately, we hope to identify the molecules of physiological significance that move between cells within the lens and to understand the implications of these movements. This study combines a cellular approach with one of a biochemical nature. In the former, cultured """"""""lentoids"""""""" will be examined since they serve as excellent models for lens fiber cells. The transfer of fluorescent dyes between cells will be monitored in a quantitative manner in order to assay for cell-to-cell channels. Changes in permeability will be analyzed following alterations: a) in intracellular pH and calcium levels, b) in cAMP levels and the activity of related protein kinases, c) in the activity of protein kinase C and d) in aspects related to cell injury. Studies with the kinases relate to the in vivo phosphorylation of the apparent lens junctional protein, MP26. Collaborative whole-cell patch-clamp experiments are included to explore various properties of the junctional channels. Membrane vesicles will be utilized to pursure channel regulation in a more defined system. The vesicles will first be characterized in order to understand the topography of MP26 and preparative conditions which influence channel activity. Since full activity for the cell-to-cell channels may not be found in the """"""""hemi-channel"""""""" form, we will also begin developing an assay for the assembly of a complete cell-to-cell channel. We believe that these studies will: a) lead to an improved understanding of the role of cell-to-cell channels in the lens and b) aid in the interpretation of different disease states in the lens (e.g., in the case of certain cataracts where junctions may not provide for a buffering of cellular differences).
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