ATP-LINKED EFFECTORS OF NA+/K+ ATPASE AND CATARACT

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Increased lens Na+ concentrations correlate both with increasing lens color and with increasing opacity in the outer fiber cell region of the lens, the lens cortex. Lens Na+ concentrations are maintained by 3 lenticular Na+ -pumps, Na,K-ATPases, which use the energy from the hydrolysis of ATP to transport K+ into lens cells and Na+ out of lens cells, presumably at a Na:K:ATP ratio of 3:2:1. In cataract lenses, ATP hydrolysis and/or K+ transport do not always change concurrently with lens Na+ concentration increases. This would suggest the Na,K-ATPase dependent Na:K transport efficiency can be altered so that ATP hydrolysis and/or K+ transport can be normal while Na+ transport is reduced. Transport efficiency may be related to gene expression (isoform function differences), to metabolite or hormone regulation, or to post- translational modification. The major long term goal of this project is to determine factors that lead to Na:K transport efficiency changes in human cataract. Na,K-ATPase function changes in other eye tissues with aging and with the same systemic diseases that correlate with human cataract. The second longterm goal is to determine if the agents that alter lenticular Na,KATPase function also cause Na,K-ATPase dysfunction in other eye tissues. The lens is unique, in that the 3 isoforms of the catalytic subunit of Na,K-ATPase, that are expressed in different regions of the lens, can be easily separated from one another by dissection. In other eye tissues mixtures of isoforms are often expressed in the same cell. Studies are proposed to characterize, by specific antibodies, catalytic subunit isoform expression in noncataractous and cataractous human lenses. The mechanism of action and Na:K transport efficiency will be determined for each lens isoform (normal and cataractous lenses) using a combination of techniques including: lens organ culture, membrane vesicle reconstitution, fusion of lens membranes into Na,K-ATPase depleted erythrocytes, Fourier Transform Infared Spectroscopy, and fluorescent substrate analog binding. Factors that affect isoform expression or that affect 150-form transport efficiency will be identified. The sites of post-translational modification of lens Na,K-ATPase isoforms by glucose, H202 and ascorbate oxidation products will be identified by sequence analysis of modified peptides isolated from proteolytic digests. Antisera to these modified peptides will be used to assess post-translational modification by these agents in human cataract.