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

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Abstract
Summary 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, Cl i and Cl o . DNDS inhibited competitively the Cl o -stimulated chloride efflux from intact red cells at 0°C and pH 7.8 with an inhibitor constant of 90nm. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5×105 molecules/cell. Cl o competitively inhibited DNDS binding with an inhibitor constant of 6mm. In the absence of Cl o the DNDS binding constant was 84mm. The competition between chloride and DNDS was also tested in nystatintreated cells in which Cl o always equaled Cl i . 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°C. We found that neither the DNDS binding constant nor the Cl o inhibitor constant were significantly changed compared to 0°C.

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