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Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes


Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for “DIDS-Band 3” complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill’s coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4’-diisothiocyanato-2,2’-stilbenedisulfonate ([3H]DIDS), the association rate constant, k+1 (M−1s−1), was measured. The mean values of (4.3 ± 0.7) × 105 M−1s−1 for control and (2.7 ± 0.7) × 105 M−1s−1 for EAC-treated ghosts were obtained. The mean values for KD, evaluated from [3H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.







1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide




tyrosine ethyl ester

WRK, Woodward’s reagent K:

N-ethyl-5-phenylisoxazolium 3’-sulfonate


  1. 1.

    Lepke, S., Heberle, J. and Passow, H. The band 3 protein: anion exchanger and anion-proton cotransporter. in: Red Cell Membrane Transport in Health and Disease (Bernhardt, I. and Ellory J.C., Eds.), Springer, Heidelberg, 2003, 221–252.

  2. 2.

    Knauf, P.A. and Pal, P. Band 3 mediated transport. in: Red Cell Membrane Transport in Health and Disease (Bernhardt, I. and Ellory J.C., Eds.), Springer, Heidelberg, 2003, 253–301.

  3. 3.

    Wieth, J.O. and Bjerrum, P.J. Titration of transport and modifier sites in the red cell anion transport system. J. Gen. Physiol. 79 (1982b) 253–282.

  4. 4.

    Zaki, L. Inhibition of anion transport across red blood cells with 1,2-cyclohexanedione. Biochim. Biophys. Acta 99 (1981) 243–251.

  5. 5.

    Wieth, J.O., Bjerrum, P.J. and Borders, C.L. Jr. Irreversible inhibition of red cell chloride exchange with phenylglyoxal, an arginine-specific reagent. J. Gen. Physiol. 79 (1982c) 283–312.

  6. 6.

    Janas, T., Janas, T., Kilian, M. and Przestalski, S. Activation energy of sulphate ion transport across methylated human erythrocyte membranes. FEBS Lett. 236 (1988) 93–94.

  7. 7.

    Wieth, J.O., Andersen, O.S., Brahm, J., Bjerrum, P.J. and Borders, C.L.Jr. Chloride-bicarbanate exchange in red blood cells. Philos. Trans. R. Soc. Lond, Series B 299 (1982a) 383–399.

  8. 8.

    Milanick, M.A. and Gunn, R.B. Proton-sulfate cotransport: mechanism of H+ and sulfate addition to the chloride transporter of human red blood cells. J. Gen. Physiol. 79 (1982) 87–113.

  9. 9.

    Milanick, M.A. and Gunn, R.B. Proton-sulfate cotransport: external proton activation of sulfate influx into human red blood cells. Am. J. Physiol. 247 (1984) C247–C259.

  10. 10.

    Cabantchik, Z. I. and Greger, G. Chemical probes for anion transporters of mammalian cell membranes. Am. J. Physiol. 262 (1992) C803–C827.

  11. 11.

    Ship, S., Shami, Y., Breuer, W. and Rothstein, A. Synthesis of tritiated 4,4’-diisothiocyano-2,2’-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells. J. Membr. Biol. 33 (1977) 311–323.

  12. 12.

    Bjerrum, P.J., Andersen, O.S., Borders, C.L.Jr. and Wieth, J.O. Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide. J. Gen. Physiol. 93 (1989) 813–839.

  13. 13.

    Funder, J., Tosteson, D.C. and Wieth, J.O. Effects of bicarbonate on lithium transport in human red cells. J. Gen. Physiol. 71 (1978) 721–746.

  14. 14.

    Funder, J. and Wieth, J.O. Chloride transport in human erythrocytes and ghosts: a quantitative comparison. J. Physiol. 262 (1976) 679–698.

  15. 15.

    Janas, T., Bjerrum, P.J., Brahm, J. and Wieth, J.O. Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes. Am. J. Physiol. 257 (1989) C601–C606.

  16. 16.

    Janas, T. and Janas, T. Reversible DIDS binding to Band 3 protein in human erythrocyte membranes. Mol. Membr. Biol. 17 (2000) 109–115.

  17. 17.

    Alder, H.L., Roessler, E.B. Introduction to probability and statistics, Freeman and Company, San Francisco, 1977.

  18. 18.

    Bjerrum, P.J. Identification and location of amino acid residues essential for anion transport in red cell membranes. in: Structure and function of membrane proteins (Quagliariello, E. and Palmiari, F., Eds.), Elsevier, Amsterdam, 1983, 107–115.

  19. 19.

    Craik, J.D. and Reithmeier, R.A.F. Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbidiimide, J. Biol. Chem. 260 (1985) 2404–2408.

  20. 20.

    Werner, P.K. and Reithmeier, R.A.F. The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl] carbodiimide. Biochim. Biophys. Acta 942 (1988) 19–32.

  21. 21.

    Miller, Ch. Ion channels: doing hard chemistry with hard ions. Curr. Opin. Chem. Biol. 4 (2000) 148–151.

  22. 22.

    Salhany, J.M., Sloan, R.L. and Cordes, K.S. The carboxyl side chain of glutamate 681 interacts with a chloride binding modifier site that allosterically modulates the dimeric conformational state of band 3 (AE1). Implications for the mechanism of anion/proton cotransport. Biochemistry 42 (2003) 1589–1602.

  23. 23.

    Jennings, M.L. Evidence for a second binding/transport site for chloride in erythrocyte anion transporter AE1 modified at glutamate 681. Biophys. J. 88 (2005) 2681–2691.

  24. 24.

    Falke, J.J. and Chan, S.I. Molecular Mechanisms of Band 3 inhibitors. 1. Transport site inhibitors. Biochemistry 25 (1986) 7888–7894.

  25. 25.

    Romero, M.L., Fulton, C.M. and Boron, W.F. The SLC4 family of HCO3 transporters. Pflugers Arch. Eur. J. Physiol. 447 (2004) 495–509.

  26. 26.

    Alper, S.L. Molecular physiology and genetics of Na+-independent SLC4 anion exchangers. J. Exp. Biol. 212 (2009) 1672–1683.

  27. 27.

    Kopito, R.R., Lee, B.S., Simmons, D.M., Lindsey, A.E., Morgans, C.W. and Schneider, K. Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59 (1989) 927–937.

  28. 28.

    Lu, J. and Boron, W.F. Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5. Am. J. Physiol. 292 (2007) C1787–C1798.

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Correspondence to Teresa Janas.

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Janas, T., Janas, T. Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes. Cell Mol Biol Lett 16, 342–358 (2011).

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Key words

  • Band 3
  • Carbodiimide
  • Dissociation constant
  • Erythrocyte membrane
  • Stilbenedisulfonate