Function and Control of A Kidney Epithelial Cell Line

Saier, Milton

Abstract

We have characterized the two predominant """"""""passive"""""""" monovalent ion transport systems present in intact kidney epithelial cells, MDCK: The amiloride-sensitive Na+/H+ antiport system (Ca++ regulated), and the furosemide-sensitive NaCl/KC1 symport system (ATP regulated). There are three closely related long range goals of the proposed research: (a) To understand the structure-function relationships of each of these two transport systems in kidney epithelial cells, MDDK, at a detailed molecular level; (b) to understand the regulatory constraints imposed upon these two systems, and (c) to understand the relationships between the function of these two ion transport systems and the regulation of cellular physiological processes (internal pH, osmolarity, membrane potential, growth). Specifically we plan: 1) To demonstrate and characterize amiloride-sensitive Na+/H+ antiport and furosemide-sensitive NaCl/KCl symport and their regulation in plasma membrane vesicles derived from MDCK cells. 2) To reconstitute both transport activities in phospholipid vesicles (liposomes) after detergent solubilization of the plasma membrane. 3) To purify and characterize the detergent solubilized proteins responsible for the transport activities from kidney tissue using the reconstitution assay. 4) To purify proteins involved in transport regulation employing either a) protein-protein binding assays, b) transport assays in plasma membrane vesicles or liposomes, or c) complementation assays employing currently available mutants defective for these two transport systems. 5) To reconstitute the regulatory mechanisms from purified components in a defined liposome system. 6) To determine the effects of hormonal agents on the activities of these two transport systems with intact cells in serum free media. 7) To use the plasma membrane vesicles to reporduce the rapid, protein synthesis-independent regulatory effects of hormones to gain evidence for or against direct protein-protein interactions or a particular second messenger mechanism. 8) To use the reconstitution system to characterize transport and regulatory proteins in mutants specifically defective in cation transport as well as in carcinogenic transformants derived from MDCK.