Mixed type inhibition of the renal Na+/H+ antiporter by the Li+ and amiloride. Evidence for a modifier site

Academic Article

Abstract

  • We studied the interactions of Na+, Li+, and amiloride on Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pH(in) = 6.0; pH(out) = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (K(m) for Li+ = 1.2 ± 0.1 mM; K(m) for Na+ = 14.3 ± 0.8 mM; V(max) for Li+ = 2.40 ± 0.07 fluorescence units/s/mg of protein; V(max) for Na+ = 7.10 ± 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], ([Na+] = 90 mM; [Li+] < 1 mM), we observed noncompetitive inhibition (V(max) for Na+/H+ exchange reduced by 25%). The apparent K(i) for this noncompetitive inhibition was ≃ 50 mM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (K(i) = 30 μM, K(i)' = 90 μM) but was only a simple competitive inhibitor of Li+/H+ exchange (K(i) = 10 μM). At [Li] = 1 mM and [amiloride] < 100 μM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.
  • Authors

    Published In

    Author List

  • Ives HE; Yee VJ; Warnock DG
  • Start Page

  • 9710
  • End Page

  • 9716
  • Volume

  • 258
  • Issue

  • 16