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 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.
Bahar
S
S
Program in Biophysics, Department of Biochemistry, University of Rochester Medical Center, Rochester New York 14642, USA. bahar@phy.duke.edu
Gunter
C T
CT
Wu
C
C
Kennedy
S D
SD
Knauf
P A
PA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid