Erythroid Band Research Articles

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.

Mutations of Conserved Arginines in the Membrane Domain of Erythroid Band 3 Lead to a Decrease in Membrane-Associated Band 3 and to the Phenotype of Hereditary Spherocytosis

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.

An Atypical Anion Transporter Functioning at Acid pH in Neuroblastoma Cells

At pH 7.4, 36Cl − uptake by neuroblastoma cells was Na +-independent, saturable and blocked by submicromolar concentrations of DIDS. This suggests that at this pH, Cl − transport is mediated by an exchanger analogous to erythroid band 3. At pH 6.2, 36Cl − uptake was markedly activated by external carboxylate anions such as acetate. Acetate-stimulated 36Cl − uptake was blocked by DIDS (IC 50 = 0.15 μM). Saturation by external 36Cl − was observed with K 0.5 = 8 mM. K 0.5 was not modified by acetate. As 36Cl efflux is also activated by acetate, we suggest the presence, in neuroblastoma cells, of an anion exchanger activated by carboxylic anions. This exchanger is active when the extracellular pH is 6.0 - 6.5.

Identification of Band 3-like Proteins and Cl-/HCO3- Exchange in Isolated Cardiomyocytes

The identification of the protein that exerts the function of Cl-/HCO3- exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl-/HCO3- exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They were specifically found in the membrane but not in the cytosolic or the myofibril fractions of adult cardiomyocytes. Confocal microscopy further indicated that the immunostained proteins were mainly located at the sarcolemma and along T-tubules, typical membrane structures of adult cardiomyocytes. Using an antibody raised against a cardiac amino-terminal domain of rat AE3, we found that the 120-kDa protein is the translation product of the AE3 gene specifically expressed in heart and brain. Using an antiserum raised against a specific domain of mouse erythroid band 3 (AE1), which is not shared by AE3, we showed that the 80-kDa protein is likely to be a truncated translation product of the AE1 gene. Microinjection of the anti-human erythroid whole band 3 antibody into single isolated cardiac cells significantly inhibited the Cl-/HCO3- exchange activity. Furthermore, the anti-AE1 antibody strongly decreased the efficiency of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to inhibit the ionic exchange. We thus suggest that the 80-kDa or both the 80- and the 120-kDa proteins immunologically related to the erythroid band 3 protein perform the anionic exchange in rat cardiomyocytes.

Anion transport function of mouse erythroid band 3 protein (AE1) does not require acylation of cysteine residue 861

Cys-861 of mouse band 3 is equivalent to Cys-843 of human band 3, the only acylated cysteine residue in the anion exchanger AE1 of the red blood cell (Hamasaki et al. (1992) Progress Cell Res. 2, 65–71). Mutation of Cys-861 to serine or methionine caused no significant changes of band 3-mediated anion exchange as measured after expression of the appropriate cRNAs in Xenopus oocytes. Susceptibility to inhibition of transport by 4,4′-dinitrostilbene-2,2′-disulfonate and PCMBS was not affected. We conclude that palmitoylation is not an absolute requirement for the successful execution the anion transport function by the hydrophobic domain of band 3 in the plasma membrane.

CDNA Sequence, Gene Sequence, and Properties of Murine Pallidin (Band 4.2), the Protein Implicated in the Murine pallid Mutation

Band 4.2, which plays an important but poorly understood role in erythrocyte function and survival, is a major component of erythrocyte membranes. Recently, it has been shown that the gene for murine protein band 4.2 colocalizes on chromosome 2 with the murine pallid mutation, which affects the formation or function of intracellular storage granules in melanocytes and platelets and lysosomes in kidney. As a first step in identifying the mutation responsible for the pallid phenotype, we have sequenced the entire normal murine band 4.2 gene. Our results show that the gene for murine band 4.2 is approximately 22 kb in size, with 13 exons and 12 intervening introns. The organization of the mouse band 4.2 gene is identical to that of the human band 4.2 gene and similar to that of the genes for the transglutaminase enzymes, reiterating the membership of protein band 4.2 in the transglutaminase gene superfamily. We also present 3.5 kb of normal murine erythroid band 4.2 cDNA sequence containing an open reading frame of 2073 bp and coding for 691 amino acids. This is the same size as the human erythrocyte protein, with which the murine protein shares a 72% amino acid identity.

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

We describe a duplication of 10 nucleotides (2,455-2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.

Distribution and function of multiple anion transporter proteins in brain tumor cell lines in relation to glucose transport

The anion transport, “band 3,” family of proteins in mammalian brain performs the same functions as that of erythroid band 3. These functions are anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. The presence of 5–7 band 3 related proteins in brain tissue was suggested by the reaction of antibodies to synthetic peptides of erythroid band 3 with a number of bands in immunoblots. Since there are a number of different cell types in brain, tissue cultures of neural cell types were examined to determine whether multiple band 3 related proteins are present in each cell type or whether several band 3 related proteins are present in each cell type. The tumor cell lines exhibit anion transport and are inhibited by the anion transport inhibitors 4,4′-diisothiocyano-2,2′ disulfonic acid (DIDS), phenylglyoxal, and furosemide. Glucose transport is inhibited by cytochalasin B and the anion transport inhibitor, phenylglyoxal, in these cell lines, but not by 4,4′-diisothiocyano-2,2′ disulfonic acid. Furosemide gave partial inhibition of most, but not all, cell lines. Since phenylglyoxal inhibits anion transport by binding to an arginine near the transport site, inhibition of glucose transport by phenylglyoxal suggests that an arginine lies in the substrate binding site. The number of cytochalasin B and DIDS binding sites was quantitated on cell lines as an approximation of the number of glucose transporter and anion transporter sites, respectively.

Band 4.1 Enhances Spectrin Binding to Phosphatidylserine Vesicles

Erythroid band 4.1 enhances the binding of erythroid spectrin to phosphatidylserine vesicles under conditions of ionic strength and at protein concentrations similar to those in the red cell. The extent of enhancement depends on the concentration of band 4.1; at 2 μM 4.1, spectrin binding increases approximately 10-fold (to 600 μmoles/mole lipid). The Kd is 0.5 μM, as measured by SDS-PAGE of protein-bound vesicles recovered by ficoll gradient centrifugation. The 4.1-enhanced binding of spectrin was also measured by a gel filtration assay. The electrostatic nature of the enhancement of spectrin binding is indicated by its dependence on the phosphatidylserine content of the vesicles.

CDNA cloning and localization of a band 3-related protein from ileum

A Cl(-)-HCO3- exchanger in the brush-border membrane mediates active Cl- absorption and regulates intracellular pH in rabbit ileum. The molecular identity of the ileal Cl(-)-HCO3- exchanger has not been established. The best-characterized plasma membrane Cl(-)-HCO3- exchanger is erythroid band 3. Structurally related proteins in nonerythroid tissues comprise an anion exchanger (AE) family. We used the polymerase chain reaction to amplify and clone a cDNA encoding an ileal band 3-related protein (B3RP) from rabbit ileal enterocytes. The composite sequence is 3,909 bp and is predicted to encode a protein of 136 kDa. The deduced amino acid sequence is 95% identical to murine renal AE2, indicating that ileal B3RP is rabbit AE2. Antisera generated against a cytoplasmic fragment of ileal B3RP recognized a 160- to 170-kDa polypeptide in the brush-border membrane, but not the basolateral membrane, of ileal crypt and villus enterocytes. This correlates with previous studies indicating that a Cl(-)-HCO3- exchange is present in brush-border but not basolateral membrane vesicles from rabbit ileal enterocytes. We conclude that ileal B3RP is a product of the AE gene family, and is present in the brush-border of ileal enterocytes, where it may mediate Cl(-)-HCO3- exchange.

A structural study of the membrane domain of band 3 by tryptic digestion. Conformational change of band 3 in situ induced by alkali treatment.

Nine peptides derived from the transmembrane domain of band 3 were purified and sequenced. All of the sequences agreed completely with deduced sequences from cDNA of human erythroid band 3. Five peptides, KS-1 to KS-5, were released from the band 3 molecule when alkali-stripped membranes were digested with trypsin, while four other peptides, KM-6 to KM-9, were obtained following subsequent urea treatment. This indicates that at least 13 new in situ cleavage sites were demonstrable by these procedures, that the released peptides are parts of hydrophilic connector loops, and that the other peptide portions constitute membrane-spanning helices. The topological designations are consistent with the hydropathy prediction of murine band 3 according to Passow ((1986) Rev. Physiol. Biochem. Pharmacol. 103, 61-203). One mol of histidine residue was found/mole of KS-1, KS-2, KS-4, and KM-6. The conformation of band 3 in situ was apparently changed by alkali treatment of erythrocyte membranes, i.e. the amount of KS-1, KS-2, and KS-4 peptides released by trypsin treatment increased as NaOH concentration was raised from 10 to 100 mM. Similarly, [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was found to bind to band 3 in membranes treated with 10 mM NaOH as well as to band 3 in white ghosts, but not to membranes treated with 100 mM NaOH. In addition, alkali treatment of membranes tended to increase the amount of band 3 cross-linked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The conformational change in band 3 by alkali treatment was also supported by the interaction of antibodies against peptides released by trypsin. The release of KS-1, KS-2, and KS-4 from the membrane was strongly inhibited by pretreating the erythrocyte membrane with DIDS, suggesting that the DIDS-band 3 complex which is in the outward facing form, is more compact and becomes resistant to trypsin compared to band 3 without DIDS.

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 crythroid 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 H 2DIDS, specific inhibitors of band 3-mediated transport. The apparent K 1 values are 3.6 μM and 0.094 μM, respectively, and hence similar to those found in the wild type band 3-mediated anion transport. The rapid reversible inhibition by H 2DIDS 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.

Role of Lys 558 and Lys 869 in substrate and inhibitor binding to the murine band 3 protein: a study of the effects of site-directed mutagenesis of the band 3 protein expressed in the oocytes of Xenopus laevis.

The effect of mutation of either Lys 558 or Lys 869 or both on mouse erythroid band 3 protein (AE1)-mediated 36Cl- efflux and its inhibition by pyridoxal 5-phosphate (P5-P), DNDS and H2DIDS were studied. Regardless of the mutation, band 3 was always capable of executing Cl- self-exchange. P5-P (5 mM, pH 7.6) produced irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues were replaced, mutant (NM), irreversible inhibition could no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues, 558 or 869. Inhibition by DNDS changed dramatically upon mutation. The Ki app increased from 6.0 microM in the wild type (KK) to 23 microM in the mutant NK, to 73 microM in the mutant KM and to 474 microM in the double mutant NM. The Km value for activation of the transport system by varying the substrate concentration by isosmotic substitution of Cl- with SO4(2-) decreased from 42 mM in the wild type (KK) to 11.3 mM in the mutant NM. The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate Cl-. In the double mutant NM, H2DIDS is no longer able to produce irreversible inhibition at pH 7.6. This is evidently related to the replacement of Lys 558 (pK 8.2) by Asn 558 in this mutant (see Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBO J. 8:3601-3609). However, at pH 9.5, some irreversible inhibition could still be observed. This suggests that the other lysine residue (pK 10.8) that is known to be involved in covalent binding with the second isothiocyanate group of H2DIDS is still present, and hence, not identical to Lys 869, which had been substituted by a methionine residue. However, this result remains inconclusive since after mutagenesis, the H2DIDS may produce inhibition at a site that is not normally involved in H2DIDS binding.

Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase

Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43 kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43 kDa protein. The 43 kDa band-3-like protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330 bp cDNA from rabbit renal cortex. Additional overlapping 272 bp and 1123 bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483 bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45,193 Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosylphosphatidylinositol. In summary, the 43 kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase.

Band 3 in Aging and Neurological Diseasea

Senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes in mammals and other vertebrates. We have created a synthetic aging antigen that blocks binding of IgG to senescent cells in vitro. Synthetic senescent cell antigen might be effective in preventing cellular destruction in vivo in certain diseases, and can be used to manipulate cellular life span in situ. Senescent cell antigen is generated by the modification of an important structural and transport membrane molecule, protein band 3. Band 3 is present in cellular, nuclear, Golgi, and mitochondrial membranes as well as in cell membranes. Band 3 proteins in nucleated cells participate in cell surface patching and capping. Band 3 maintains acid-base balance by mediating the exchange of anions (e.g., chloride, bicarbonate), and is the binding site for glycolytic enzymes. It is responsible for CO2 exchange in all tissues and organs. Thus, it is the most heavily used anion transport system in the body. Band 3 is a major transmembrane structural protein which attaches the plasma membrane to the internal cell cytoskeleton by binding to band 2.1 (ankyrin). Oxidation generates senescent cell antigen in situ. Band 3 is present in the central nervous system, and differences have been described in band 3 between young and aging brain tissue. One autosomal recessive neurological disease, choreoacanthocytosis, is associated with band 3 abnormalities. The 150 residues of the carboxyl terminus segment of band 3 appear to be altered. In brains from Alzheimer's disease patients, antibodies to aged band 3 label the amyloid core of classical plaques and the microglial cells located in the middle of the plaque in tissue sections, and an abnormal band 3 in immunoblots. Band 3 protein(s) in mammalian brain performs the same functions as that of erythroid band 3. These functions is anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. Structural similarity of brain and erythroid band 3 is suggested by the reaction of antibodies to synthetic peptides of erythroid band 3 with brain band 3, the inhibition of anion transport by the same inhibitors, and an equal degree of inhibition of brain and erythrocyte anion transport by synthetic peptides of erythroid band 3. One of these segments, pep-COOH, contains antigenic determinants of senescent cell antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain membrane protein band 3 performs the same functions as erythrocyte band 3.

We report the presence of band 3 protein(s) in mammalian brain that performs the same functions as those of erythroid band 3. These functions are anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. Structural similarity of brain and erythroid band 3 is suggested by the reaction of antibodies to synthetic peptides of erythroid band 3 with brain band 3, the inhibition of anion transport by the same inhibitors, and an equal degree of inhibition of brain and erythrocyte anion transport by synthetic peptides of erythroid band 3 (pep-ANION 2, residues 588-602; pep-COOH, residues 812-827; pep-COOH-N6, residues 813-818). One of these segments, pep-COOH, contains antigenic determinants of senescent cell antigen. These findings suggest that the transport domains of erythroid and neural band 3 are similar functionally and structurally and support the hypothesis that the immunological mechanism of maintaining homeostasis is a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates.

PH-dependence of inhibition by H 2DIDS 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′-disulfonate (H 2DIDS) 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 p K value of Lys a is about 2.5 p K units lower than the p K value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H 2DIDS 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 H 2DIDS at a concentration (20 μM) which saturates all H 2DIDS binding sites with reversibly bound H 2DIDS ( K I = 0.3 μM and 1.1. μM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H 2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H 2DIDS 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 H 2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, p K 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 H 2DIDS 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 p K 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 p K of 10.8. These values are 0.8–0.9 p K 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). The difference can be accounted for by the difference of temperature which amounted to 20°C in the present experiments with the oocytes and to 30°C in the previous work with red blood cells. The results suggest that location and function of Lys a and Lys b are essentially the same after expression in the lipid bilayer of the red blood cell and the oocyte.

<i>Drosophila</i> to Bacteriophage to Erythrocyte: The Erythrocyte as a Model for Molecular and Membrane Aging of Terminally Differentiated Cells

Senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes in mammals and other vertebrates. Although the initial studies are done using erythrocytes as a model, senescent cell antigen has been found on all cells examined. Oxidation generates senescent cell antigen in situ. Senescent cell antigen is generated by the modification of an important structural and transport membrane molecule, protein band 3. Band 3 is a ubiquitous protein. It is present in cell, nuclear, Golgi, and mitochondrial membranes. Band 3 is the most heavily used anion exchanger in the human body because of its crucial role in respiration and acid-base balance. Senescent cell antigen has been localized to band 3 residues 538-554 and 812-827, using competitive inhibition studies with synthetic peptides of band 3 to absorb the IgG isolated from senescent erythrocytes and immunoblotting studies. In mammalian brain, band 3 performs the same functions as that of erythroid band 3. These functions are anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. Our results suggest that the transport domain of erythroid and neural band 3 are similar functionally and structural. This supports the hypothesis that the immunological mechanism of maintaining homeostasis is a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates.

Effects of membrane potential on electrically silent transport. Potential-independent translocation and asymmetric potential-dependent substrate binding to the red blood cell anion exchange protein.

Tracer anion exchange flux measurements have been carried out in human red blood cells with the membrane potential clamped at various values with gramicidin. The goal of the study was to determine the effect of membrane potential on the anion translocation and binding events in the catalytic cycle for exchange. The conditions were arranged such that most of the transporters were recruited into the same configuration (inward-facing or outward-facing, depending on the direction of the Cl- gradient). We found that the membrane potential has no detectable effect on the anion translocation event, measured as 36Cl(-)-Cl- or 36Cl(-)-HCO3- exchange. The lack of effect of potential is in agreement with previous studies on red cells and is different from the behavior of the mouse erythroid band 3 gene expressed in frog oocytes (Grygorczyk, R., W. Schwarz, and H. Passow. 1987. J. Membr. Biol. 99:127-136). A negative potential decreases the potency of extracellular SO4= as an inhibitor of either Cl- or HCO3- influx. Because of the potential-dependent inhibition by SO4=, conditions could be found in which a negative intracellular potential actually accelerates 36Cl- influx. This effect is observed only in media containing multivalent anions. The simplest interpretation of the effect is that the negative potential lowers the inhibitory potency of the multivalent anion by lowering its local concentration near the transport site. The magnitude of the effect is consistent with the idea that the anions move through 10-15% of the transmembrane potential between the extracellular medium and the outward-facing transport site. In contrast to its effect on extracellular substrate binding, there is no detectable effect of membrane potential on the competition between intracellular Cl- and SO4= for transport sites. The lack of effect of potential on intracellular substrate binding suggests that the access pathway leading to the inward-facing transport site is of lower electrical resistance than that leading to the extracellular substrate site.

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.