Robert B. Gunn

Altered structure and anion transport properties of band 3 ( AE1, SLC4A1 ) in human red cells lacking glycophorin A

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (MkMk cells) or GPA (En(a–) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to ∼60% of that in normal control red cells as a result of an increased apparent Km for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the Vmax for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal Kd for iodide binding, in agreement with the unchanged Km found in transport studies. However, the apparent diffusion quenching constant (Kq) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in Vmax observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.

Anion Transport in Sickle Red Blood Cells

ABSTRACT: Anion transport in sickle cells (SS RBC) mediated by the band 3 membrane protein was evaluated by three different measures in both oxygenated and deoxygenated conditions and compared to normal red cells. First, Cl- self-exchange measured as 36Cl- efflux at 0°C was normal in SS RBC in both Vmax and dependence on extracellular Cl- concentration. There was no effect of deoxygenation on either parameter. Second, stilbene-sensitive 35SO=4:SO=4exchange, measured at 37°C where morphologic sickling occurred, was also unaffected by deoxygenation and was normal compared to normal red cells. Third, conductive Cl- flux was assessed by measuring the rates of Cl−-limited K+ efflux in valinomycin-treated cells at 37°C. Both the stilbene-sensitive and insensitive components of net Cl- flux were similar in SS RBC and normal red cells, and were unaltered by morphologic sickling. Thus, despite dramatic alterations in cation transport in SS RBC and the demonstration of interaction between band 3 protein and sickle cell, anion transport functions appear to be normal in SS RBC and are unaffected by deoxygenation. These data suggest that the majority of the anion exchangers in SS RBC are functionally normal.