American Journal of Physiology -- Legacy Content 1999-10-01

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

S Bahar, C T Gunter, C Wu, S D Kennedy, P A Knauf

Index: Am. J. Physiol. 277(4 Pt 1) , C791-9, (1999)

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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 by (35)Cl-NMR show that external Cl(-) binds to band 3 even when Cl(-) transport is inhibited approximately 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.