Transport-related conformational states of the band 3 protein: probing with 1-fluoro-2,4-dinitrobenzene.

The present article provides experimental evidence for previous claims, that Lys 539, without being directly involved in anion binding or translocation, is allosterically linked to the anion binding sites of the band 3 protein and to some other, as yet unidentified amino acid residue. The evidence is based on a detailed study of the kinetics of inhibition of sulphate equilibrium exchange by 1-fluoro-2,4-dinitrobenzene (N2ph-F). It is shown that the mutation of Lys 558 in mouse band 3, which is homologous to Lys 539 in human band 3, renders the transport protein insusceptible to inhibition by N2pH-F, confirming that it is the modification of this residue which results in the inhibition of band 3-mediated transport. The investigation of the kinetics of the modification of human band 3 revealed that the modification is not preceded by non-covalent N2ph-F binding and hence governed by the structure of the native protein near Lys 539. In chloride-containing media, the rate constant of dinitrophenylation of Lys 539 is about 15 times higher than in sulphate-containing media. This suggests that the chemical nature of the anion species bound to band 3 determines whether Lys 539 exists in a buried or exposed state and hence represents a reporter group which characterizes the functional state of the transport protein. The parameter values describing the effects of anion binding on the interactions between Lys 539 and an allosterically linked, unidentified amino acid residue were determined by means of a mathematical model which permitted the quantitative evaluation of the data.

Inhibition of mouse erythroid band 3-mediated chloride transport by site-directed mutagenesis of histidine residues and its reversal by second site mutation of Lys 558, the locus of covalent H2DIDS binding.

Substitution by site-directed mutagenesis of any one of the histidine residues H721, H837, and H852 by glutamine, or of H752 by serine, inhibits Cl- flux mediated by band 3 expressed in Xenopus oocytes. Mutation of Lys 558 (K558N), the site of covalent binding of H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) in the outer membrane surface, in combination with any one of the His/Gln mutations leads to partial (H721Q; H837Q) or complete (H852Q) restoration of Cl- flux. In contrast, inhibition of Cl- flux by mutation of proline or lysine residues in the vicinity of His 837 at the inner membrane surface cannot be reversed by the second-site mutation K558N, indicating specificity of interaction between Lys 558 and His 837. The histidine-specific reagent diethyl pyrocarbonate (DEPC) is known to inhibit band 3-mediated anion exchange in red blood cells [Izuhara, K., Okubo, K., & Hamasaki, N. (1989) Biochemistry 28, 4725-4728]. It was also found to inhibit transport after expression in the oocyte of wild-type band 3, of the double mutants of the histidines listed above, and of the single mutant H752S. The effects on the wild type and the double mutants were indistinguishable, while the mutant H752S exhibited a considerably reduced sensitivity to inhibition, suggesting that His 752 is the most prominent site of action of DEPC. According to a hydrophobicity plot of band 3 and further independent evidence, Lys 558, the mutated histidines, and Glu 699, the mutation of which was also found to inhibit Cl- flux [Müller-Berger, S., Karbach, D., Kang, D., Aranibar, N., Wood, P. G., Rüterjans, H., & Passow, H. (1995) Biochemistry 34, 9325-9332], are most likely located in five different transmembrane helices. The interactions between Lys 558 and the various histidines suggest that these helices reside in close proximity. Together with the helix carrying Glu 699, they could form an access channel lined with an array of alternating histidine and glutamate residues. Together with a chloride ion bridging the gap between His 852 and His 837, they could have the potential to form, at low pH, a transmembrane chain of hydrogen bonds. The possible functional significance of such channel is discussed.

Mediation of inorganic anion transport by the hydrophobic domain of mouse erythroid band 3 protein expressed in oocytes of Xenopus laevis.

A cDNA clone of the mouse erythroid band 3 protein encoding the 556 amino acid residues of the hydrophobic domain from Thr-374 to the C-terminal Val-929 is shown by immunoprecipitation to be expressed in Xenopus oocytes. Measurements of 36Cl- efflux indicate that the translation product mediates Cl- transport, which is inhibitable reversibly by DNDS or H2DIDS, specific inhibitors of band 3-mediated transport. The apparent KI values are 3.6 microM and 0.094 microM, respectively, and hence similar to those found in the wild type band 3-mediated anion transport. The rapid reversible inhibition by H2DIDS slowly changes to irreversible inhibition. The rate of change increases with increasing pH, again similar as to the wild-type band 3. It is concluded that the hydrophobic domain of band 3 is capable of executing anion transport essentially similar to the full-length band 3, although minor differences with respect to transport and inhibition kinetics cannot be ruled out.

pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue.

The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4'-diisothiocyanodihydrostilbene-2,2'-disfulfonate (H2DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b. In the red cell membrane, the pK value of Lys a is about 2.5 pK units lower than the pK value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H2DIDS far exceeds the susceptibility to Lys b. In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl- exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H2DIDS at a concentration (20 microM) which saturates all H2DIDS binding sites with reversibly bound H2DIDS (KI = 0.3 microM and 1.1 microM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H2DIDS from the extracellular phase. Subsequent measurements of Cl- efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, pK values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H2DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single pK value of 8.3. In the mutant, Lys a is missing. Hence, covalent reaction can only take place with Lys b. Measurements over the appropriate pH range could be described by a single pK of 10.8. These values are 0.8-0.9 pK units higher than those previously obtained in experiments with band 3 in the red cell membrane (Kampmann et al. (1982) J. Membr. Biol. 70, 199-216).(ABSTRACT TRUNCATED AT 250 WORDS)

Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein--encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site.

A vector was constructed containing a cDNA for mouse band 3 obtained from Demuth et al. (1986, EMBO J., 5, 1205-1214), a synthetic linker (containing 5'-non-translated region, start codon and a coding region for the first 12 N-terminal amino acids), and RNA polymerase promoters suitable for in vitro transcription of cRNA. After injection of the cRNA into the cytoplasm of Xenopus oocytes and incubation for 16 h, expression of mouse band 3 was demonstrated by immunoprecipitation, immunohistochemical methods and influx or efflux measurements with 36Cl-. Antisense cRNA inhibits the expression. Lysines 558 and 561 were replaced by asparagines using oligonucleotide-directed mutagenesis. Like the original band 3, the mutant shows stilbene disulfonate-inhibitable anion exchange. However, in contrast to the original band 3, inhibition by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate (H2DIDS) is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 involves one of the two modified lysine residues. It also shows that the two lysine residues are not essential for the execution of the anion transport function of band 3. The results described suggest that the cDNA clone of Demuth et al. (1986) encodes a protein with properties that are representative for the properties of the bulk of the band 3 protein in the plasma membrane of the red cell of the mouse.

Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H2DIDS binding site of the band 3 protein.

The inhibition of anion transport by dinitrophenylation of the red cell membrane is brought about by the modification of a single lysine residue located on the 17-kDa segment of the band 3 protein. This residue is identical with Lys a, which is also capable of reacting with one of the two isothiocyanate groups of 4,4'-diisothiocyano dihydro-stilbene-2,2'-disulfonate (H2DIDS). The rate of reaction between Lys a and 1-fluoro-2,4-dinitrobenzene is reduced when a second lysine residue on the 35-kDa segment of the band 3 protein becomes dinitrophenylated. This latter residue is not identical with Lys b which is known to be present on the 35-kDa segment and involved in the cross-linking of this segment with the 17-kDa segment by H2DIDS.

Inverse effects of dansylation of red blood cell membrane on band 3 protein-mediated transport of sulphate and chloride.

1. Dansylation of the red cell membrane produces inverse effects on SO(4) (2-) and Cl(-) equilibrium exchange. The former is enhanced by several orders of magnitude (Legrum, Fasold & Passow, 1980), the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulphophenyl)-6-methyl-7-benzothiazol sulphonic acid (APMB), a disulphonic acid that combines non-covalently with the 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulphonic acid (H(2)DIDS) binding site of the anion transport protein.2. After dansylation the maximum of the pH dependence of SO(4) (2-) exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level at around pH 7.0 and resembles that observed by Funder & Wieth (1976) for Cl(-).3. The mutual interactions between the transfer site, the H(2)DIDS binding site, and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H(2)DIDS binding site by the non-covalently binding agents 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS), 4,4'-bis(acetamido) stilbene-2,2'-disulphonate (DAS) or APMB inhibit the enhanced SO(4) (2-) exchange across the previously dansylated membrane. The apparent K(I) value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS, and to 1/60 for APMB. Conversely, when dansylation is carried out while the H(2)DIDS binding site is occupied by DNDS, APMB or DAS, the enhancement of SO(4) (2-) exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. This suggests that the agents stabilize different conformations of the H(2)DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites.4. The SO(4) (2-) equilibrium exchange as measured at a fixed Cl(-) concentration is enhanced when the Cl(-) concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites.5. The enhanced K(+) efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.

The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid (H2DIDS).

The two isothiocyanate groups of the anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2-2'-disulfonate (H2DIDS) may react covalently with two lysine residues called a and b that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction leads to the establishment of intramolecular cross-links between a and b (M.L. Jennings & H. Passow, 1979, Biochim. Biophys. Acta 554:498-519). In the present work, the time course of the reactions with a and b, and of the establishment of the cross-link were investigated experimentally and compared with simple mathematical models of the reaction sequence. The rates of reaction with a and b were found to increase with increasing pH. Regardless of pH, the rate of reaction with a exceeds that with b several-fold. Once the H2DIDS molecule has reacted with a, the rate of the subsequent reaction of the other isothiocyanate group with b is reduced by about 1/30. The reactions that follow the unilateral attachment to site b are not yet clear. A more detailed analysis of the time course of the cross-linking reaction suggests that a satisfactory description of the kinetics requires the assumption that the H2DIDS binding site may exist in two different states, and that the transition from one state to the other is associated with changes of the reactivities of either lys a alone or of both lys a and b. This led to the formulation of the two-states model of the H2DIDS binding site, which is supported by other pieces of independent evidence. The analysis of the pH dependence of the rate of thiocyanylation of b shows that the apparent pK value of that lysine residue is about 9.9 to 10.0 and hence slightly lower than the intrinsic pK of a lysine residue in an aqueous environment.

Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts.

Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 degrees C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1-19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 mug/ml. After digestion at 25 mug/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourth peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[35S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentration exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulfonic acid, 4,4'-diacetamido stilbene-2,2'-disulfonic acid, and 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS).

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

The permeability of the human red blood cell to sulfate ions.

Sulfate permeability was measured at Donnan equilibrium as a function of three variables, the sulfate, chloride, and hydrogen ion concentration in the medium. The data were used for a quantitative evaluation of a number of typical predictions of a fixed charge model of the ion permeable regions of the red blood cell membrane. It could be shown that more than 1,000-fold variations of sulfate flux,[Formula: see text], could be represented as a function of a single variable SO 4m (2-) , the sulfate concentration in the membrane. SO 4m (2-) was calculated from the measured values of all three variables by means of a previously published equation (Passow,Progress in Biophysics and Molecular Biology, vol. 19, pt. II, pp. 425-467, 1969). In this equation, two constants can be arbitrarily chosen:A, the sum of the charged and uncharged forms of dissociable fixed charges, andK, the dissociation constant of the fixed charges. For the present calculations, the previously obtained valuesA=2.5 andK=1·10(-9) were used. The resulting relationship between[Formula: see text] and SO 4m (2-) was found to obey the equation[Formula: see text] wherec I=1.62·10(-9),c II=2.3·10(-2),a=4.94 gave the best fit for data obtained at 27°C. The exponential increase of[Formula: see text] with SO 4m (2-) suggests that there exists a cooperative facilitation of sulfate flux with increasing SO 4m (2-) . Measurements of the apparant activation energy of sulfate flux yielded a value of 32.7 Kcal/mole. This value was independent of the pH at which the measurements were made.