INCREASED NA+-H+ ANTIPORTER ACTIVITY IN APICAL MEMBRANE-VESICLES FROM MUTANT LLC-PK1 CELLS

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na+-H+ antiporter activity at the apical surface (J. G. Haggerty, N. Agarwal, R. F. Reilly, E. A. Adelberg, and C. W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, Na-22 influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na+-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na+-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na+-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na+-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na+-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na+-H+ antiporter. The availability of membrane vesicle preparations with pharmacologically different Na+-H+ antiporters provides a model system for further biochemical characterization of these two types of transporters.