The LLC-PK, cell line, which was originally derived from pig kidney, is a widely used model system for studying the structure and physiological properties of renal transporters. During the past grant period, we have discovered that LLC-PKI cells express two distinct forms of the Na/H antiporter: NAH-1, found in rapidly growing cells and on the basolateral surface at confluence, and NAH-2, appearing on the apical surface at confluence. The two forms exhibit a 300-fold difference in sensitivity to ethylisopropylamiloride and are also affected independently by genetic mutations. During the next grant period, we will pursue several main goals. Starting with the NAH-1 gene, which we have already cloned and sequenced, we will attempt to isolate the gene for NAH-2 by low-stringency hybridization. Alternative strategies have been designed in the event that this straightforward approach is not successful. As soon as both genes are in hand, specific DNA probes and antibodies will be developed to track the synthesis and localization of NAH-1 and NAH-2 during differentiation, both in the normal LLC-PK,/Clone 4 cell line and in two existing mutants. Additional antiporter-deficient mutants will be isolated and characterized to obtain information about structure-function relationships. And finally, the cloned NAH-1 and NAH-2 genes will be expressed in antiporter-deficient cells and/or in yeast to confirm the physiological properties of the two antiporters and to pave the way for site-directed mutagenesis. Taken together, the results of this work should fumish useful new information about the structure, biogenesis, and transport mechanism of Na/H antiporters in the kidney.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037279-07
Application #
3292559
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1986-07-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Reilly, R F; Hildebrandt, F; Biemesderfer, D et al. (1991) cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells. Am J Physiol 261:F1088-94
Igarashi, P; Reilly, R F; Hildebrandt, F et al. (1991) Molecular biology of renal Na(+)-H+ exchangers. Kidney Int Suppl 33:S84-9
Reilly, R F; Haggerty, J G; Aronson, P S et al. (1991) Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells. Am J Physiol 260:C738-44
Huot, S J; Cassel, D; Igarashi, P et al. (1989) Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger. J Biol Chem 264:683-6
Haggerty, J G; Agarwal, N; Reilly, R F et al. (1988) Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1). Proc Natl Acad Sci U S A 85:6797-801
Haggerty, J G; Agarwal, N; Cragoe Jr, E J et al. (1988) LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride. Am J Physiol 255:C495-501