Substrate-dependent reversal of anion transport site orientation in the human red blood cell anion-exchange protein, AE1

The tightly coupled, one-for-one exchange of anions mediated by the human red blood cell AE1 anion-exchange protein involves a ping-pong mechanism, in which AE1 alternates between a state with the anion-binding site facing inward toward the cytoplasm (Ei) and a state with the site facing outward toward the external medium (Eo). The conformational shift (Ei <----> Eo) is only permitted when a suitable substrate such as Cl- or HCO(3) over bar (B-) is bound. With no anions bound, or with Cl- bound, far more AE1 molecules are in the inward-facing than the outward-facing forms (Ei much greater than Eo, ECli much greater than EClo). We have constructed a model for Cl--B- exchange based on Cl--Cl- and B--B- exchange data, and have used it to predict the heteroexchange flux under extremely asymmetric conditions, with either all Cl- inside and all B- outside (Cli-Bo) or vice versa (Bi-Clo). The experimental values of the ratio of the exchange rate for Bi-Clo to that for Cli-Bo are only compatible with the model if the asymmetry of bicarbonate-loaded sites (A(B) = EBo/EBi) > 10, the opposite of the asymmetry for unloaded or Cl-loaded sites. Furthermore, the Eo form has a higher affinity for HCO(3) over bar than for Cl-, whereas the Ei form has a higher affinity for Cl-. Thefact that this "passive" system exhibits changes in substrate selectivity with site orientation ("sidedness"), a characteristic usually associated with energy-coupled "active" pumps, suggests that changes in affinity with changes in sidedness are a more general property of transport proteins than previously thought.