The purpose of this project is to describe the mechanisms of membrane transport in ocular tissues at the cellular and molecular level. Alterations of these processes are directly related to disease states such as corneal disfunction, retinal detachments and cataracts. The experimental approach will consist of: 1) Studies of chloride channels in corneal epithelial cell membranes of the bovine and frog eyes, 2) Vesicular transport in cellular apical or basolateral membranes of the corneal epithelium, retina pigment epithelium and lens fiber membranes of bovine eyes 3) Characterization of membrane proteins involved in the transport process, such as the Na-K-2Cl cotransporter and band 3 analogs in the membranes of epithelia and fibers. The chloride channels of the apical membranes will be examined by the use of membrane vesicles of apical preparations of corneal epithelial cell of bovines and frogs during their fusion to an artificial lipid bilayer. Channel analysis will permit study of their properties and action of specific activators or inhibitors of the channel function. The transport studies in membrane vesicles obtained from ocular epithelia and lens fiber membranes will investigate Na/H exchanger, D-glucose and Na-K-2Cl cotransporters proteins in corneal epithelium, retinal pigment epithelium and lens fiber membranes of b vine. The vesicular transport studies permit the kinetic characterization of functional membrane proteins. This leads to the next step which is the chemical characterization of these proteins.
This aim will be accomplished by SDS-PAGE of membranes; binding to specific inhibitors such as stilbene, either fluorescent or radiolabelled, and binding to loop diuretics like radiolabelled furosemide or bumetanide. The purification of these proteins will be followed by sequencing in order to understand their primary structure. The results of the work will increase the knowledge of basic mechanisms of membrane protein function associated to derrangment of corneal transparency retinal attachment or detachment and the development of lens cataracts.
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