Vol. 277, Issue 4, C791-C799, October 1999
Persistence of external chloride and DIDS binding after
chemical modification of Glu-681 in human band 3
Sonya
Bahar,
Christopher T.
Gunter,
Cheryl
Wu,
Scott D.
Kennedy, and
Philip A.
Knauf
Program in Biophysics, Department of Biochemistry and
Biophysics, University of Rochester Medical Center, Rochester New
York 14642
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
35Cl-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.
anion exchangers; anion transport; red blood cells; erythrocytes; nuclear magnetic resonance; anion exchanger 1
