scholarly journals Transmembrane effects of irreversible inhibitors of anion transport in red blood cells. Evidence for mobile transport sites.

1979 ◽  
Vol 73 (4) ◽  
pp. 493-514 ◽  
Author(s):  
S Grinstein ◽  
L McCulloch ◽  
A Rothstein
Keyword(s):  
Mobile Element ◽  
Band 3 ◽  
Anion Transport ◽  
Permeability Barrier ◽  
Irreversible Inhibitor ◽  
Red Cell Membrane ◽  
Intact Cells ◽  
High Concentrations ◽  
Cytoplasmic Side ◽  
Transport Site

Experiments were designed to determine whether band 3, the anion transport protein of the red cell membrane, contains a mobile element that acts as a carrier to move the anions across a permeability barrier. The transport site-specific, nonpenetrating irreversible inhibitor 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) was found to be effective only when applied extracellularly. It was used to sequester transport sites on the extracellular side of the membrane in intact cells. The membranes were then coverted into inside-out vesicles. The number of anion transport sites available on the cytoplasmic side of the vesicle membranes was then estimated by measuring the binding of N-(-4-azido-2-nitrophenyl)-2-aminoethyl-sulfonate (NAP-taurine), a photoreactive probe. Pretreatment with DIDS from the extracullular side substantially reduced the binding of NAP-taurine at the cytoplasmic side. Since NAP-taurine does not appear to penetrate into the intravesicular (normally extracellular) space, a transmembrane effect is apparently involved. About 70% of the DIDS-sensitive NAP-taurine binding sites are located in band 3, with the remainder largely in a lower molecular weight (band 4) region. A similar pattern of reduction in NAP-taurine binding is produced by high concentrations of Cl-, but this anion has little or no effect in vesicles from cells pretreated with DIDS. Thus the DIDS-modulated sites seem to be capable of binding either NAP-taurine or Cl. It is suggested that band 3 contains a mobile transport element that can be recruited to the extracellular surface by DIDS, thus becoming unavailable to NAP-taurine at the cytoplasmic face of the membrane. The results are consistent with a model of carrier-mediated transport in which the movement of the transport site is associated with a local conformational change in band 3 protein.

Inhibition of chloride binding to the anion transport site by diethylpyrocarbonate modification of band 3

1990 ◽  
Vol 116 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Naotaka Hamasaki ◽  
Kenji Izuhara ◽  
Kenshi Okubo ◽  
Yoko Kanazawa ◽  
Akira Omachi ◽  
...  
Keyword(s):  
Band 3 ◽  
Anion Transport ◽  
Chloride Binding ◽  
Transport Site

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

1986 ◽  
Vol 250 (6) ◽  
pp. C955-C969 ◽  
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.

scholarly journals Monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein. Localization of the C-terminus of the protein to the cytoplasmic side of the red cell membrane and distribution of the protein in some human tissues

1989 ◽  
Vol 258 (1) ◽  
pp. 211-220 ◽  
Author(s):  
S D Wainwright ◽  
M J A Tanner ◽  
G E M Martin ◽  
J E Yendle ◽  
C Holmes
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Human Erythrocyte ◽  
Anion Transport ◽  
Transport Protein ◽  
Red Cell ◽  
Membrane Domain ◽  
Red Cell Membrane ◽  
C Terminus ◽  
Cytoplasmic Side

(1) We have prepared murine monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein (band 3). (2) All of these antibodies react with regions of the protein located at the cytoplasmic surface of the red cell. (3) One of the antibodies reacts with an epitope present on a cytoplasmic loop of the protein located between the C-terminus and a point 168 amino acids from the C-terminus. The other antibodies recognize different epitopes on the C-terminal tail of the protein and the sequences likely to be involved in these epitopes are defined. (4) Our results show that the C-terminus of the red-cell anion transport protein is located on the cytoplasmic side of the red-cell membrane. (5) None of the antibodies inhibited sulphate exchange transport when introduced into resealed red-cell membranes; however, the bivalent form of one of the antibodies reduced the inhibitory potency of 4-acetamido-4'-isothiocyanatostilbene disulphonate on sulphate exchange transport in resealed erythrocyte membranes. (6) Immunostaining of human kidney sections with the antibodies showed strong staining of the basolateral membrane of some but not all of the epithelial cells of distal tubules and the initial connecting segment of collecting tubules. With human liver, only the haematopoeitic cells of fetal liver reacted with all the antibodies.

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

1989 ◽  
Vol 257 (2) ◽  
pp. C277-C289 ◽  
Author(s):  
P. A. Knauf ◽  
L. J. Spinelli ◽  
N. A. Mann
Keyword(s):  
Conformational Changes ◽  
External Medium ◽  
Band 3 ◽  
Anion Transport ◽  
Flufenamic Acid ◽  
Band 3 Protein ◽  
Inhibitory Potency ◽  
External Site ◽  
Transport Molecules ◽  
Transport Site

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.

scholarly journals Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

1983 ◽  
Vol 81 (4) ◽  
pp. 453-484 ◽  
Author(s):  
P J Bjerrum ◽  
J O Wieth ◽  
C L Borders
Keyword(s):  
Intracellular Ph ◽  
Covalent Binding ◽  
Chloride Concentration ◽  
Protein Band ◽  
Band 3 ◽  
Anion Transport ◽  
Transport Protein ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Arginyl Residues

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.

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