Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system.

Abstract

Niflumic acid is a noncompetitive inhibitor of Cl exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl-, the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the Cl gradient is reversed, with Clo > Cli, the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. The intrinsic dissociation constants for Cl- at the 2 sides of the membrane are nearly equal. Thus, the Cl-loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with .apprx. 15 times more sites facing the inside than the outside. The asymmetry reflects an .apprx. 1.5 kcal/mol free energy difference between the inward-facing and outward-facing Cl-loaded forms of band 3. High concentrations of Cl (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that Cl binds equally well to the modifier site regardless of the orientation of the transport site.