AJP - Renal Physiology, Vol 261, Issue 6 1088-F1094, Copyright © 1991 by American Physiological Society

ARTICLES

cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells

R. F. Reilly, F. Hildebrandt, D. Biemesderfer, C. Sardet, J. Pouyssegur, P. S. Aronson, C. W. Slayman and P. Igarashi
Department of Internal Medicine, Yale University, School of Medicine, New Haven, Connecticut 06510.

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na(+)-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na(+)-H+ exchanger. There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na(+)-K(+)-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na(+)-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.

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