Inhibition of anion permeability by amphiphilic compounds in human red cell: Evidence for an interaction of niflumic acid with the band 3 protein

Abstract

In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I ₅₀=6×10⁻⁷ m) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts shows one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4′-isothiocyano stilbene-2,2′-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9×10⁵ per cell) agrees with the number of protein molecules per cell in band 3. These sites are halfsaturated with 10⁻⁶ m niflumic acid, a concentration very close toI ₅₀. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3. Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.