Evidence for a second binding/transport site for chloride in erythrocyte anion transporter AE1 modified at glutamate 681

Abstract

Transport kinetics have been examined in erythrocyte anion transporter AE1 that has been chemically modified to convert glutamate 681 to an alcohol (E681OH AE1). Outward conductive Cl-; flux in E681OH AE1 is inhibited by removal of extracellular Cl-; this effect is the opposite of that in native AE1 and is consistent with coupled electrogenic 2:1 Cl -/Cl- exchange. A second Cl- binding/transport site is also suggested by the characteristics of 35SO 42- flux in E681OH AE1: bilateral and cis Cl-, which are normally inhibitory, accelerate 35SO42- flux. These effects would be expected if Cl- binds to a second transport site on SO42--loaded E681OH AE1, thereby allowing Cl-/SO42- cotransport. Alternatively, the data can be explained without proposing Cl-/SO42- cotransport if the rate-limiting event for 36SO 42-/SO42- exchange is external SO42- release, and the binding of external Cl- accelerates SO42- release. With either interpretation, these data indicate that E681OH AE1 has a binding/transport site for Cl - that is distinct from the main transport site. The effects of graded modification of E681 or inhibition by H2DIDS are consistent with the idea that the new Cl- binding site is on the same E681OH-modified subunit of the AE1 dimeras the normal transport site. © 2005 by the Biophysical Society.