Flufenamic acid senses conformation and asymmetry of human erythrocyte band 3 anion transport protein

With Cl as substrate, the human red blood cell anion transport (band 3) protein can exist in four conformations: Ei, with the transport site facing the cytoplasm; Eo, with the transport site facing the external medium; and ECli and EClo, the corresponding forms loaded with Cl. Flufenamic acid (FA), an inhibitor that binds to an external site different from the transport site, binds to Eo with a dissociation constant of 0.0826 +/- 0.0049 (SE) microM. Binding of iodide or sulfate to the external-facing transport site reduces the affinity by 1.66 or 14.3-fold, respectively. Changing from Eo to Ei lowers the affinity by 3.7-fold, and binding of cytoplasmic iodide to Ei further decreases the affinity by 5.5-fold. Thus changes in orientation of the transport site and substrate binding, even at the opposite side of the membrane, cause sufficient conformational changes in band 3 to affect FA binding substantially. If the possible effects of Cl binding to the transport site on FA affinity are estimated from the iodide data, the dependence of FA inhibitory potency on Cl concentrations inside and outside the cell suggests that there are at least 6.5 times as many inward-facing as outward-facing Cl-loaded transport sites. This information can be used to calculate the distribution of capnophorin among the various conformations under different circumstances and to devise conditions for recruiting the transport molecules toward a particular conformation.

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Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.6.c955 ◽

1986 ◽

Vol 250 (6) ◽

pp. C955-C969 ◽

Cited By ~ 17

Author(s):

M. A. Milanick ◽

R. B. Gunn

Keyword(s):

Conformational Changes ◽

Chloride Concentration ◽

Band 3 ◽

Anion Transport ◽

Band 3 Protein ◽

Chloride Flux ◽

Proton Binding ◽

Chloride Exchange ◽

Transport Site ◽

External Ph

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.

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