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

Abstract

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)(-) (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 Eo, ECli EClo). We have constructed a model for CI(-)-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)(-) than for Cl(-), whereas the Ei form has a higher affinity for Cl(-). The fact 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.

Fig 1.

Efflux of ³⁶Cl⁻ from red blood cells into 150 mM HCO medium (150 KHCO₃) or of H¹⁴CO into 150 mM Cl⁻ medium (150 KH). Mean values for [Cli] and for [HCO₃i] are given in Materials and Methods and in Fig. 2. Because the internal anion content is nearly the same in all experiments, rate constants (k) are proportional to the fluxes (J). Solid line and black triangles, Bi-Clo, k = 0.199 ± 0.015 s⁻¹; dashed line and black circles, Cli-Bo, k = 0.0336 ± 0.0003 s⁻¹; dotted line and white triangles, Bi-Clo with 10 μM DIDS (4,4′-diisothiocyano-stilbene-2,2′-disulfonate) added to the flux medium, k = 0.0079 ± 0.0003 s⁻¹.

Fig 2.

Calculation of the heteroexchange fluxes with Cl⁻ inside and HCO outside (J_Cli-Bo, dashed line) or HCO inside and Cl⁻ outside (J_Bi-Clo, solid line). The fluxes were calculated (12, 13) for various values of A_B (= EBo/EBi), with the self-consistent model parameters from Table 1, so that the maximum Cli-Clo and Bi-Bo fluxes would be consistent with experimental data (7). The mean values for [Cli] (116 ± 2 mM, SEM, n = 4) and [HCO₃i] (112 ± 4 mM, n = 3) from the experiments were used, and A_Cl (= ECli/EClo) was 0.14 (S. D. Kennedy, C. A. Wu, and P.A.K., unpublished data; see text for details).

Fig 3.

Ratio of HCO efflux with Bi-Clo to Cl⁻ efflux with Cli-Bo (solid line). Ratios were determined as a function of A_B from calculated fluxes in Fig. 2. Dash-dot line shows value (1.3) expected for ratio if A_B is the same as A_Cl, 0.14. Dashed line shows mean of efflux rate constant values from experiments as in Fig. 1, with dotted lines indicating ±1 SEM.

Fig 4.

Rationale for asymmetry in Cli-Bo and Bi-Clo fluxes. The majority forms of AE1 are shown in bold type: Ei ≫ Eo, ECli ≫ EClo, and EBo ≫ EBi. The asymmetries for Cl⁻ and HCO–loaded sites imply that the rate constants for transitions between these forms must be asymmetric, with the solid line indicating the faster rate constant in each case. For the clockwise cycle, Bi-Clo exchange, the faster steps are involved for both bicarbonate and chloride transport across the membrane (black solid lines), but for the anticlockwise cycle, Cli-Bo exchange, the slower steps are used (dashed lines).