scholarly journals 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.

1984 ◽  
Vol 83 (5) ◽  
pp. 657-681 ◽  
Author(s):  
W Furuya ◽  
T Tarshis ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Competitive Inhibitor ◽  
Transport Protein ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Anion Exchange Protein ◽  
Ping Pong ◽  
Chloride Exchange ◽  
Transport Site

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.

scholarly journals Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry.

1984 ◽  
Vol 83 (5) ◽  
pp. 683-701 ◽  
Author(s):  
P A Knauf ◽  
F Y Law ◽  
T Tarshis ◽  
W Furuya
Keyword(s):  
Anion Exchange ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Disulfonic Acid ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Chloride Exchange ◽  
A Site ◽  
Transport Site

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.

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

1984 ◽  
Vol 83 (5) ◽  
pp. 703-725 ◽  
Author(s):  
P A Knauf ◽  
N A Mann
Keyword(s):  
Anion Exchange ◽  
Dissociation Constants ◽  
Energy Difference ◽  
Protein Band ◽  
Band 3 ◽  
Niflumic Acid ◽  
Inhibitory Potency ◽  
The Asymmetry ◽  
High Concentrations ◽  
Transport Site

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.

Anion exchange protein (band 3) in human erythrocytes characterized by different abnormalities

1993 ◽  
Vol 220 (2) ◽  
pp. 211-217 ◽  
Author(s):  
A. Giuliani ◽  
S. Marini ◽  
L. Ferroni ◽  
S.G. Condo' ◽  
M.T. Ramacci ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Anion Exchange Protein ◽  
Exchange Protein

scholarly journals Flexibility of the Cytoplasmic Domain of the Anion Exchange Protein, Band 3, in Human Erythrocytes

2001 ◽  
Vol 81 (6) ◽  
pp. 3363-3376 ◽  
Author(s):  
Scott M. Blackman ◽  
Eric J. Hustedt ◽  
Charles E. Cobb ◽  
Albert H. Beth
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Anion Exchange Protein ◽  
Exchange Protein

Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation

1993 ◽  
Vol 264 (5) ◽  
pp. C1144-C1154 ◽  
Author(s):  
P. A. Knauf ◽  
N. M. Strong ◽  
J. Penikas ◽  
R. B. Wheeler ◽  
S. Q. Liu
Keyword(s):  
Inhibitory Concentration ◽  
Potent Inhibitor ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Band 3 ◽  
Reversible Inhibitor ◽  
Red Cell ◽  
Inhibitory Potency ◽  
A Site ◽  
Transport Site

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

NIP- and NAP-taurine bind to external modifier site of AE1 (band 3), at which iodide inhibits anion exchange

1995 ◽  
Vol 269 (2) ◽  
pp. C410-C416 ◽  
Author(s):  
P. A. Knauf ◽  
L. J. Spinelli
Keyword(s):  
Anion Exchange ◽  
Blood Cells ◽  
Inhibitory Effect ◽  
Band 3 ◽  
Reaction Site ◽  
Inhibitory Potency ◽  
Human Red Blood Cells ◽  
Inhibitory Site ◽  
Probable Reaction ◽  
Transport Site

External iodide (I-o) inhibits AE1 (band 3)-mediated anion exchange in human red blood cells by binding to a noncompetitive inhibitory site, the external halide modifier site. External N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) and N-(4-isothiocyano-2-nitrophenyl)-2-aminoethyl sulfonate (NIP-taurine) also inhibit Cl- exchange noncompetitively. Increasing I-o decreases the inhibitory potency of NIP-taurine in a competitive fashion; this effect is not due to I- binding to the transport site, which has little effect on the NIP-taurine affinity. Bis(sulfosuccinimidyl)-suberate (BSSS) abolishes the noncompetitive inhibitory effect of I-o and greatly reduces the inhibitory effect of NAP-taurine. Together with previous work, these data suggest that external halides, such as I-, Br-, and probably also Cl-, bind to the same noncompetitive inhibitory site as do NAP- and NIP-taurine and that these reagents can be used to label the halide modifier site. Lys-539, a probable reaction site of BSSS, lies within the same segment of AE1 that is labeled by NAP-taurine and thus may be part of the modifier site.

Kinetics of DIDS inhibition of HL-60 cell anion exchange rules out ping-pong model with slippage

1991 ◽  
Vol 260 (3) ◽  
pp. C535-C544 ◽  
Author(s):  
D. Restrepo ◽  
B. L. Cronise ◽  
R. B. Snyder ◽  
L. J. Spinelli ◽  
P. A. Knauf
Keyword(s):  
Anion Exchange ◽  
Competitive Inhibition ◽  
Disulfonic Acid ◽  
Inhibition Kinetics ◽  
Ping Pong ◽  
Kinetics Of ◽  
Simultaneous Model ◽  
Substrate Kinetics ◽  
Transport Site ◽  
Rule Out

According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.

scholarly journals Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein.

1996 ◽  
Vol 107 (2) ◽  
pp. 293-306 ◽  
Author(s):  
S Q Liu ◽  
E Ries ◽  
P A Knauf
Keyword(s):  
Dissociation Constant ◽  
Competitive Inhibitor ◽  
Anion Exchange Protein ◽  
Or Groups ◽  
External Site ◽  
Selective Reagent ◽  
Guanidino Group ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.

Sign in / Sign up

Export Citation Format

Share Document