The external anion binding site of the human erythrocyte anion transporter: DNDS binding and competition with chloride

Abstract

The interaction between chloride and the anion transport inhibitor DNDS (4,4'-dinitro stilbene-2,2'-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cli and Clo. DNDS inhibited competitively the Clo-stimulated chloride efflux from intact red cells at 0 degree C and pH 7.8 with an inhibitor constant of 90 nM. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5 X 10(5) molecules/cell. Clo competitively inhibited DNDS binding with an inhibitor constant of 6 mM. In the absence of Clo the DNDS binding constant was 84 mM. The competition between chloride and DNDS was also tested in nystatintreated cells in which Clo always equaled Cli. Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20 degree C. We found that neither the DNDS binding constant nor the Clo inhibitor constant were significantly changed compared to 0 degree C.