The cornea is the primary refractive element of the eye and therefore it must be smooth and transparent for good vision. Transparency is most affected by the hydration level of the stromal connective tissue. Hydration is dependent on the metabolic activities of the corneal epithelium and endothelium, however it is the endothelium that is primarily responsible for pumping ions and fluid that maintains the steady-state hydration of the stroma. The long-term goal of this project is to understand how the corneal endothelium does this job, so that medical therapies could be developed to boost endothelial function in corneas that have been compromised by disease or trauma. The endothelium is most dependent on HCO3- for its function. A number of cell membrane HCO3- related transport mechanisms have been identified and characterized on the basolateral surface (facing the stroma) of the endothelium. Our focus in this application is to establish the roles of some prominent transport mechanisms for HCO3- and fluid transport, including: 1) the basolateral Na+: HCO3- co-transporter, 2) the apical membrane bound carbonic anhydrase (CA-IV), 3) the anion exchanger and 4) the Cystic Fibrosis Transmembrane Regulator (CFTR). To establish these roles, HCO3- fluxes and the ability of the cornea to maintain its thickness (i.e., hydration) will be measured under conditions where a transport mechanism is inhibited by pharmacological, proteolytic or antisense knockdown approaches. The locations of these transport proteins will be confirmed by immunofluorescence and membrane protein separation techniques. The role of CA-IV in CO2/HCO3- transport is most intriguing. The function of CA-IV will be probed more deeply by measuring the effect of its activity on the apical surface pH. We propose to construct a novel surface pH probe using pH sensitive Green Fluorescent Protein (GFP) that is directed to the apical membrane and tethered to the extracellular surface by a glycosylphosphatidylinositol (GPI) anchor. Lastly, we will use RT-PCR and sequencing to identify new HCO3- related transport proteins, then characterize their function and determine if their expression, e.g., some anion exchangers, is dependent on the presence of CFTR.
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