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

Abstract

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward’s reagent K ( N-ethyl-5-phenylisoxazolium-3′-sulfonate; WRK). Measurements of Cl− binding by35Cl-NMR show that external Cl− binds to band 3 even when Cl− transport is inhibited ∼95% by WRK modification of Glu-681. This indicates that the external Cl− binding 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 Glu-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 facing Cl−transport site are located nearer to the external surface of the membrane than Glu-681.