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

Abstract

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.