Increased Na+-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells

R. F. Reilly, J. G. Haggerty, P. S. Aronson, E. A. Adelberg, C. W. Slayman

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3 Scopus citations

Abstract

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 γ-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na 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 α-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.

Original languageEnglish (US)
Pages (from-to)C738-C744
JournalAmerican Journal of Physiology - Cell Physiology
Volume260
Issue number4 29-4
DOIs
StatePublished - 1991

Keywords

  • Epithelial cell line
  • Intracellular pH
  • Sodium

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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