Eye lens transparency depends to a great extent on the vectorial transport of metabolites, waste products and water between the anterior and posterior lens surfaces. Fiber cells, the principal component of mammalian lens, impose unique demands upon transport; they lack tight junctions, the structures that regulate transport through the extracellular clefts, and contain only small concentrations of Na,K- ATPase, the enzyme that maintains the cell's resting potential by moving Na+ and K+ against their electrochemical gradients. The principal aim of this application is to study the structure and function of the channel for communication between fiber cells in calf lenses (the cell-to-cell channel). The channels form a pathway which is principally responsible for the diffusion of metabolites, waste products and water throughout fiber cells. The application proposes to purify the fiber cell-to-cell channels from bovine calf lenses using non-ionic detergents and column chromatography methods. Experiments are proposed to determine whether fiber cell-to-cell channels are assembled from one connexin (homotypic) or two (i.e., a """"""""mixed"""""""" channel). Purified cell-to-cell channels will be crystallized in two-dimensional sheets and their structure studied by cryo-electron microscopy and computer image processing. The purified channels also will be reconstituted in planar lipid bilayers and characterized electrically at the single and multichannel levels. Another aim of the application is to study the pathways of specialized regions of the lens called sutures which are formed by the interaction of fiber cells from opposite regions of the lens. These sutures are important in maintaining lens transparency because their extracellular clefts constitute a direct pathway into the lens interior. Sutures also contain large number of membranous vesicles and tubules which may function in the transport of molecules into fiber cells via a pinocytotic mechanism. The proposed studies of the structure and function of fiber cell-to-cell channels and sutures will be important for the understanding of the mechanisms involved in the maintenance of lens transparency and in the formation of cataracts, the principal disease of the lens.
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