Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3

Although its primary function is monovalent anion exchange, the band 3 prot ein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conse rved throughout the anion exchanger family:(AE family). To determine whethe r or not the monovalent anion binding site is located near Glu-681, we modi fied this residue with Woodward's reagent K (N-ethyl-5-phenyl-isoxazolium-3 '-sulfonate; WRK). Measurements of Cl- binding by Cl-35-NMR show that exter nal Cl- binds to band 3 even when Cl- transport is inhibited similar to 95% by WRK modification of Glu-681. This indicates that the external Cl- bindi ng site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl- binding even when WRK is bound to G lu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facin g Cl- transport site are-located nearer to the external surface of the memb rane than Glu-681.