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, 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Reilly
R F
RF
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Haggerty
J G
JG
Aronson
P S
PS
Adelberg
E A
EA
Slayman
C W
CW
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002999:Clone Cells; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D009154:Mutation; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; D005723:gamma-Glutamyltransferase; D000254:Sodium-Potassium-Exchanging ATPase; C039614:ethylisopropylamiloride