LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride.

Abstract

LLC-PK1 cells contain a well-characterized Na+-H+ antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al. (J. Biol. Chem. 261: 14614-14620, 1986), we have selected mutants that can recover from an acid load in the presence of 100 microM EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (Vmax) for the Na+-H+ antiporter, assayed as EIPA-sensitive 22Na+ uptake, has increased from 44 nmol.min-1.10(6) cells-1 in the parent cells to 106 nmol.min-1.10(6) cells-1 in PKE20. No detectable change has occurred in the Km for Na+ (118 mM in the parent, 111 mM in the mutant) or in the dependence of Na+ uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na+-K+ pump and Na+-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na+ concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.