In this proposal, an approach to the study of the regulation of the plasma membrane Na+-H+ antiporter is outlined. The experimental model employed is a clone (clone 4) of an established pig kidney cell line, the LLC-PK1 cell line. The principle methodology involves the use of a fluorescent probe that is sensitive to changes in intracellular pH (pHi). The probe, 2,7-biscarboxyethyl-5(6)-carboxyfluoroscein (BCECF) is well suited for measuring changes in pHi since its pKa is approximately 7.0. With the aid of the probe it is possible to study Na+-H+ antiport activity by monitoring changes in pHi.
The specific aims of this proposal are a) to characterize the Na+-H+ antiporter in LLC-PK1-Cl4 cells, b) to identify the role of H+ in modulating antiport activity at the internal locus in the intact cell, c) to define a role for intracellular Na+ (Nai) in modifying antiport activity, d) to test whether growth promoting factors and mitogens augment antiport activity and particularly e) to define the interaction between growth factors, mitogens, H+, and Na+ at the internal locus of the exchanger. The data gained from this project will provide important information regarding the regulation of transport pathway that is intricately linked to important cellular processes. Long-term goals include the study of the cascade of events leading up to activation of the antiporter.
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