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 Cl. > 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 = Cl. 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 chlorideloaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with -15 times more sites facing the inside than the outside. The asymmetry reflects an -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 Cl i = 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 .
Experiments were performed with intact human red blood cells to determine whether the inhibitory effects of high Cl- concentrations on Cl- exchange are primarily due to interaction at the cytoplasmic or the external surface of the membrane. When internal Cl- was varied from 150 mM to 600 mM Cl- (using the nystatin technique), keeping external Cl- constant at 150 mM (with sucrose added to maintain osmotic balance), Cl- exchange was inhibited almost exactly as much as when both internal and external Cl- were increased from 150 mM to 600 mM. On the other hand, if internal Cl- was maintained constant at 600 mM, variation of external Cl- (with either sucrose, gluconate, or citrate-sucrose mixtures replacing Cl-) had no consistent effect on Cl- exchange. Even if internal Cl- was kept at 150 mM by substitution of gluconate for Cl-, an increase in external Cl- from 150 mM to 600 mM did not significantly inhibit Cl- exchange. Thus the self-inhibitory effects of Cl- seem to be caused primarily by binding to a site at the cytoplasmic side of the membrane. External Br-, on the other hand, did cause a significant inhibition of Cl- exchange. In contrast to the inhibitory effects of Cl- at neutral pH, at very high pH (around pH 11) there is an activation of Cl- exchange at very high Cl- concentrations. This effect, however, depends on binding of Cl- to an external site. Thus there seem to be at least two different low-affinity Cl- binding sites, one at the cytoplasmic side, which inhibits Cl- exchange, and one at the external side, which activates Cl- exchange at high external pH.
N-(4-isothiocyano-2-nitrophenyl)-2-aminoethanesulfonate (NIP-taurine), a newly synthesized isothiocyano derivative of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate (NAP-taurine), is a potent inhibitor of human erythrocyte chloride exchange. At 0 degrees C, the inhibition is reversible, but at 37 degrees C, NIP-taurine inhibits irreversibly, indicating that it may be a useful label for its binding site. When present at the outside of the cell, NIP-taurine binds with low affinity to a site that seems to be the Cl- transport site (on the basis of its affinity for Cl-) and with much higher affinity to a different site, MN, which has a much lower affinity for Cl-. In this respect, NIP-taurine resembles NAP-taurine, and an analysis of interactions between these two probes is consistent with the idea that they bind to the same two sites. The affinity of NIP-taurine for the high-affinity MN site is enhanced by about fourfold when the transport protein, band 3, is in the conformation with the transport site facing outward (Eo), as compared with the conformation with the transport site facing inward (Ei). External Cl-, but not cytoplasmic Cl-, competes with NIP-taurine for binding to the external, high affinity site. Thus NIP-taurine provides a label for an external site, at which Cl- and perhaps other anions bind, which is separate from both the transport site and the cytoplasmic modifier site at which high Cl- concentrations inhibit Cl- exchange.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.