In human senile cataracts and in most animal models of cataractogenesis, cataract formation is associated with increases in both sodium and calcium inside lens fiber cells. Because both sodium and calcium are charged molecules, it is expected that their movements across lens cell membranes will depend upon the transmembrane potential across those membranes. Potassium channels are known to be important in setting the magnitude of the transmembrane potential and thus to indirectly affect the movements of both sodium and calcium. In this proposal, molecular biology, electrophysiology, and high resolution optical imaging methods are applied to epithelial and fiber cells from lenses of several species of animals. It is the intent of the proposed experiments to identify at a molecular level, all of the potassium channels that are important contributors to the resting voltage of the lens. This includes determining their primary amino acid sequence, cloning their genes, and expressing the genes in mammalian expression systems so as to characterize each in the absence of the other potassium channels which the lens possesses. In addition, by constructing fusion proteins of the channels with green fluorescence protein, localization of the channels in two natural lens preparations will be determined as well as obtaining estimates of the time required by these cells for expression of the proteins. Localization of the messenger RNA to several regions of fiber cells will also be done. It is anticipated that the information collected from these experiments will identify candidate potassium channels which might be associated with the sodium and calcium changes which occur during cataractogenesis.
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