Functions of extracellular lysine residues in the human erythrocyte anion transport protein.
The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D002352:Carrier Proteins / Q000502:physiology*; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008239:Lysine / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D010446:Peptide Fragments; D013388:Succinimides
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D010446:Peptide Fragments; D013388:Succinimides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C035760:bis(sulfosuccinimidyl)suberate; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid