The external anion binding site of the human erythrocyte anion transporter: DNDS binding and competition with chloride

Summary The interaction between chloride and the anion transport inhibitor DNDS (4,4-dinitro stilbene-2,2-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cl _i and Cl _o . DNDS inhibited competitively the Cl _o -stimulated chloride efflux from intact red cells at 0°C and pH 7.8 with an inhibitor constant of 90nm. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5×10⁵ molecules/cell. Cl _o competitively inhibited DNDS binding with an inhibitor constant of 6mm. In the absence of Cl _o the DNDS binding constant was 84mm. The competition between chloride and DNDS was also tested in nystatintreated cells in which Cl _o always equaled Cl _i . Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20°C. We found that neither the DNDS binding constant nor the Cl _o inhibitor constant were significantly changed compared to 0°C.     

Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl- , the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride- loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.     

Fluorescence labeling of the human erythrocyte anion transport system.

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419--2427). The vesicles have a functional anion transprot system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescnece titrations show a maximum labeling stoichiometry of 1.3 +/- 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu2+ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion transport system. Sulfate transport in the labeled vesicles remains fully functional. We detected self-energy transfer between bound FMA molecules by fluorescence depolarization. With excitation at 450--50 nm P decreases from 0.4, when less than half of the proteins are labeled, to 0.1 at saturation. This depolarization is not observed with red edge excitation (510--530 nm). Addition of 0.1% sodium dodecyl sulfate (SDS) changes P to 0.32, regardless of the excitation wavelength or degree of saturation with FMA. These results indicate that the band 3 proteins are close enough to allow energy transfer between fluorophores(Ro = 37.4 A), which does not occur upon red edge excitation or when the proteins are separated by SDS. We conclude that the functional anion transport system exists as a dimer or higher oligomer of band 3 proteins in these membranes, confirming previous suggestions derived using other methods. Future applications are discussed.     

Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.     

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

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 (35)SO(4)(2-) flux in E681OH AE1: bilateral and cis Cl(-), which are normally inhibitory, accelerate (35)SO(4)(2-) flux. These effects would be expected if Cl(-) binds to a second transport site on SO(4)(2-)-loaded E681OH AE1, thereby allowing Cl(-)/SO(4)(2-) cotransport. Alternatively, the data can be explained without proposing Cl(-)/SO(4)(2-) cotransport if the rate-limiting event for (35)SO(4)(2-)/SO(4)(2-) exchange is external SO(4)(2-) release, and the binding of external Cl(-) accelerates SO(4)(2-) 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 H(2)DIDS are consistent with the idea that the new Cl(-) binding site is on the same E681OH-modified subunit of the AE1 dimer as the normal transport site.     

Functional characterization of anion transport system isolated from human erythrocyte membranes.

The anion transport system of human red blood cells was isolated in vesicles containing the original lipids of the membrane and predominantly the 95,000-dalton polypeptides (Band 3) associated with intralipid particles. The vesicles display various characteristic properties of anion permeation closely resembling those of the native system. The properties include energy of activation, pH dependence, anion sleectivity, sensitivity to specific inhibitors, and exchange and net rates of sulfate transport. Based on these and other criteria, the functional properties of isolated vesicles could be equated with those of the intact cell system. Direct support for the involvement of 95,000-dalton polypeptides in permeation functions is provided.     

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

Mapping the ankyrin-binding site of the human erythrocyte anion exchanger

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

Normalization of receptor binding of apolipoprotein E2. Evidence for modulation of the binding site conformation

Apolipoprotein (apo-) E3, when combined with the phospholipid dimyristoylphosphatidylcholine (DMPC), binds avidly to apo-B,E (low density lipoprotein) receptors on human fibroblasts. Apolipoprotein E2 isolated from type III hyperlipoproteinemic subjects, which differs from apo-E3 by the presence of cysteine instead of arginine at residue 158, possesses only about 1% of the receptor binding activity of apo-E3. Modification of apo-E2 with cysteamine, which converts the cysteine at position 158 to a positively charged lysine analogue, activates receptor binding approximately 13-fold. In the present experiments, thrombin was used to cleave apo-E2 into two fragments (Mr = 22,000 and Mr = 10,000). The larger fragment, which has been shown to possess the receptor binding domain, displayed binding activity up to 12-fold greater than intact apo-E2 or equivalent to apo-E2 treated with cysteamine. When the Mr = 22,000 fragment was modified with cysteamine and combined with DMPC, receptor binding was further enhanced, attaining the level of activity of normal apo-E3 X DMPC, a 100-fold increase over apo-E2 X DMPC binding. When the cysteamine modification was reversed by incubation with beta-mercaptoethanol, the Mr = 22,000 fragment retained most of its binding activity. However, when the same sample was tested 24 h later, the level of binding activity dropped significantly. The receptor binding of apo-E2-containing beta-very low density lipoproteins could also be activated by cysteamine treatment, with the same retention of enhanced binding activity occurring after the reversal of the modification. These results indicate that apo-E2 can attain full binding activity by the removal of the carboxyl-terminal one-third of the molecule and the addition of a positive charge at residue 158 of the molecule. The retention of enhanced binding after the reversal of the cysteamine modification indicates that the enhanced binding is probably due to conformational changes induced in the binding domain (and maintained by the phospholipid) and not merely to the presence of the positive charge at residue 158.     

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.     

Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min- 1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.