Red Blood Cell Anion Exchange Research Articles

Molecular Simulations Reveal the Dynamics of the Band 3 Anion Transporter in a Model Native Red Blood Cell Membrane

Band 3, the red blood cell anion exchanger (AE1/SLC4A1), is responsible for the rapid transport of bicarbonate and chloride across the red blood cell plasma membrane, a process necessary for efficient respiration. Human Band 3 is comprised of a cytosolic domain and a membrane domain that contains 14 transmembrane helices. Although structural data are available for both isolated domains, the structure of the complete Band 3 remains elusive. This is a limiting factor in the study of Band 3 anion transport and of its interactions with cytosolic proteins. By integrating molecular modelling and molecular dynamics simulations at the coarse-grained and all-atom resolutions, we have constructed a model of Band 3 that consists of both the transmembrane and the cytosolic domains. This model enabled us to identify the orientation of the cytosolic domain relative to the transmembrane domain and the role of the linker regions that connect these two domains in their interactions. Our model was validated using functional data and molecular simulations. Simulations were performed in complex bilayers that resemble the native red cell plasma membrane, containing a full complement of phospholipids, sphingomyelin and cholesterol. Our results provide novel molecular insights into the interactions of the lipid environment with Band 3. Specific lipids, e.g. cholesterol, have been found to localize in the Band 3 dimer interface possibly stabilizing the dimer. Specific lipid head groups were also found to interact with the cytosolic domain regulating its orientation relative to the transmembrane domain. Moreover, our modeling approach identified key residues involved in anion binding. Understanding the dynamics and interactions of Band 3 in a model native red cell membrane provides new insights into the function of this important human transporter.

Cell membrane surface expression and tyrosine kinase regulate the osmolyte channel (skAE1) in skate erythrocytes

A regulatory volume decrease response occurs when skate (Raja erinacea) erythrocytes are exposed to hyposmotic medium; they swell and then reduce their volume by releasing organic osmolytes (primarily taurine) and associated water. The response involves the red blood cell anion-exchanger skate anion-exchanger 1 (skAE1), which has been previously shown to be affected by tyrosine phosphorylation, to form tetramers and to change binding affinities to the cytoskeletal proteins, ankyrin and band 4.1. Our recent studies are focused on determining the sequence and mechanism of these events to better understand the activation of skAE1 upon hyposmotic stimulation. Under isosmotic conditions a large portion of skAE1 is found not only on the plasma membranes but also associated intracellularly in detergent-resistant membranes or lipid rafts. We hypothesize that an important step in the hyposmotic-induced increase in taurine transport involves the movement of skAE1 from an intracellular association with lipid rafts into the cell membrane. Inhibition of tyrosine phosphorylation of skAE1 with piceatannol reduces the hyposmotic-induced increase in taurine transport and also inhibits both the decreased binding of skAE1 to band 4.1 and the increased affinity to ankyrin. However, the phosphorylation inhibitor does not block the movement of the transporter into the plasma membrane or the formation of tetramers. This suggests that tyrosine phosphorylation is important in the hyposmotic-induced taurine transport but other steps that do not require phosphorylation play an important role.

Tyrosine kinase inhibition affects skate anion exchanger isoform I alterations after volume expansion

Upon exposure to hypotonic medium, skate red blood cells swell and then reduce their volume by releasing organic osmolytes and associated water. The regulatory volume decrease is inhibited by stilbenes and anion exchange inhibitors, suggesting involvement of the red blood cell anion exchanger skAE1. To determine the role of tyrosine phosphorylation, red blood cells were volume expanded with and without prior treatment with the tyrosine kinase inhibitor piceatannol. At the concentration used, 130 microM, piceatannol nearly completely inhibits p72(syk), a tyrosine kinase previously shown to phosphorylate skAE1 (M. W. Musch, E. H. Hubert, and L. Goldstein. J Biol Chem 274: 7923-7928, 1999). Hyposmotic-induced volume expansion stimulated association of p72(syk) with a light membrane fraction of skate red blood cells. Piceatannol did not inhibit this association but decreased hyposmotically stimulated increased skAE1 tyrosine phophorylation. Movement of skAE1 from an intracellular to a surface detergent-resistant membrane domain and tetramer formation were not inhibited by piceatannol treatment. Two effects of hyposmotic-induced volume expansion, decreased band 4.1 binding and increased ankyrin, were both inhibited by piceatannol. These results suggest that at least one event requiring p72(syk) activation is pivotal for hyposmotic-induced increased transport; however, steps that do not require tyrosine phosphorylation may also play a role.

Buffering limits plasma HCO3− dehydration when red blood cell anion exchange is inhibited

Theory suggests that HCO3- dehydration in the plasma of rainbow trout is limited by both the absence of carbonic anhydrase (CA) activity and the low non-bicarbonate buffer capacity of the plasma (betaplasma). The potential for betaplasma to limit plasma HCO3- dehydration was assessed in rainbow trout in which HCO3- dehydration via the red blood cell (RBC) was inhibited using the anion exchange blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). DIDS administration reduced the rate of RBC HCO3- dehydration by 68-80% for at least 6h, resulting in the elevation of arterial CO2 tension (PaCO2) by 3.07 +/- 0.45 Torr (N = 6). Addition of bovine CA to the circulation of DIDS-treated trout caused PaCO2 to decrease significantly. This effect was increased significantly in rainbow trout in which betaplasma was elevated experimentally by intravascular injection of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), supporting the hypothesis that CA-catalysed HCO3- dehydration in the plasma of rainbow trout is limited by proton availability.

Multiple transport functions of a red blood cell anion exchanger, tAE1: its role in cell volume regulation.

1. It was previously shown that expressed in Xenopus oocyte the mouse (mAE1) and the trout (tAE1) anion exchanger behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with a chloride channel activity. The present data, obtained by measurement of Xenopus oocyte membrane permeability and conductance, provide evidence that tAE1 also induces a large increase in Na(+) and K(+) permeability inhibited by several AE1 inhibitors. 2. This inhibition does not result from an effect on the driving force for electrodiffusion but represents a direct effect on the cation pathway. 3. As a control, expression of cystic fibrosis transmembrane conductance regulator (CFTR) having, once stimulated by 3-isobutyl-1-methylxanthine (IBMX), the same anion conductance magnitude as tAE1 did not induce any cation movement. 4. Chloride exchange, channel activity and cation transport induced by anion exchanger expression are inhibited by free or covalently bound H2DIDS as well. This covalent inhibition is reversed by the point mutation of Lys-522, the covalent binding site of H2DIDS to the protein. These data reveal that tAE1 itself acts both as an anion exchanger and as a channel of broad selectivity. 5. All results obtained by expression of AE1 isoforms in Xenopus oocytes and those obtained in erythrocytes are consistent with the proposal that, in nucleated erythrocytes, tAE1 functions as the swelling-activated osmolyte anion channel involved in cell volume regulation. In contrast AE1 from mammalian red cells, which do not regulate their volume, lacks swelling-activated osmolyte channel properties. 6. tAE1 illustrates the ability of a specific transport system to be a multifunctional protein exhibiting other transport functions when submitted to regulation.

Acute and chronic influence of temperature on red blood cell anion exchange.

Unidirectional (36)Cl(-) efflux via the red blood cell anion exchanger was measured under Cl(-) self-exchange conditions (i.e. no net flow of anions) in rainbow trout Oncorhynchus mykiss and red-eared freshwater turtle Trachemys scripta to examine the effects of acute temperature changes and acclimation temperature on this process. We also evaluated the possible adaptation of anion exchange to different temperature regimes by including our previously published data on other animals. An acute temperature increase caused a significant increase in the rate constant (k) for unidirectional Cl(-) efflux in rainbow trout and freshwater turtle. After 3 weeks of temperature acclimation, 5 degrees C-acclimated rainbow trout showed only marginally higher Cl(-) transport rates than 15 degrees C-acclimated trout when compared at the same temperature. Apparent activation energies for red blood cell Cl(-) exchange in trout and turtle were lower than values reported in endothermic animals. The Q(10) for red blood cell anion exchange was 2.0 in trout and 2.3 in turtle, values close to those for CO(2) excretion, suggesting that, in ectothermic animals, the temperature sensitivity of band-3-mediated anion exchange matches the temperature sensitivity of CO(2) transport (where red blood cell Cl(-)/HCO(3)(-) exchange is a rate-limiting step). In endotherms, such as man and chicken, Q(10) values for red blood cell anion exchange are considerably higher but are no obstacle to CO(2) transport, because body temperature is normally kept constant at values at which anion exchange rates are high. When compared at constant temperature, red blood cell Cl(-) permeability shows large differences among species (trout, carp, eel, cod, turtle, alligator, chicken and man). Cl(-) permeabilities are, however, remarkable similar when compared at preferred body temperatures, suggesting an appropriate evolutionary adaptation of red blood cell anion exchange function to the different thermal niches occupied by animals.

Abnormal Erythrocyte Anion Exchange in Alzheimer Disease

Several abnormalities have been described in red blood cells of patients with Alzheimer disease (AD), but to date none of these has been confirmed by a second, independent study. Erythrocyte anion exchange has been reported to be abnormal in AD; we have developed a new technique for measuring anion exchange. To confirm the abnormality of erythrocyte anion exchange in AD and to determine whether the phenomenon has potential for clinical utility. Comparison of patients with probable AD to age-matched controls. University hospital and ambulatory clinic. Chloride-bicarbonate exchange was measured in erythrocyte ghosts resealed with a fluorescent probe of chloride concentration. Erythrocyte anion exchange is abnormal in AD. This difference appears in citrate but not EDTA anticoagulant. Mahalanobis's generalized distance between the 2 populations is 1.7, and a discriminant function derived from our technique classifies 82% of the study population in accordance with the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria. Receiver operating characteristic analysis demonstrates the possibility of choosing cutoffs with high sensitivity and specificity. Measurement of red blood cell anion exchange may be useful in classifying patients with AD. The dependence of this phenomenon on anticoagulant suggests the involvement of platelet activation or complement fixation.

Carbon dioxide transport and red blood cell anion exchange function in alligators and other reptiles

Carbon dioxide transport and red blood cell anion exchange function in alligators and other reptiles

Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

The structure of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene

The structure and sequence of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene was determined by analysis of genomic and cDNA clones. The gene extends over 18 kb and consists of 20 exons. The cDNA sequence comprises 4,906 nucleotides [excluding the poly(A) tail]. There is extensive similarity between the human and mouse AE1 gene, although the latter covers 17 kb. The additional sequence present within the human AE1 gene compared with the mouse gene is mainly caused by the presence of 6 Alu repetitive units in the human gene between intron 13 and exon 20, one of which is within the 3′ noncoding region of the cDNA. Two potential promoter regions within the human AE1 gene are located so that they could give rise to the different transcripts found in erythroid cells and in the kidney. The kidney transcript would lack exons 1 through 3 of the erythroid transcript. The translation initiator downstream to the human kidney promoter would give rise to a protein with a 20 amino acid section at the N-terminus that is not present in the erythroid human AE1 protein or the rodent kidney AE1 proteins.

The Structure of the Human Red Blood Cell Anion Exchanger (EPB3, AE1, Band 3) Gene

The structure and sequence of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene was determined by analysis of genomic and cDNA clones. The gene extends over 18 kb and consists of 20 exons. The cDNA sequence comprises 4,906 nucleotides [excluding the poly(A) tail]. There is extensive similarity between the human and mouse AE1 gene, although the latter covers 17 kb. The additional sequence present within the human AE1 gene compared with the mouse gene is mainly caused by the presence of 6 Alu repetitive units in the human gene between intron 13 and exon 20, one of which is within the 3' noncoding region of the cDNA. Two potential promoter regions within the human AE1 gene are located so that they could give rise to the different transcripts found in erythroid cells and in the kidney. The kidney transcript would lack exons 1 through 3 of the erythroid transcript. The translation initiator downstream to the human kidney promoter would give rise to a protein with a 20 amino acid section at the N-terminus that is not present in the erythroid human AE1 protein or the rodent kidney AE1 proteins.

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin Ulex europaeus agglutinin I

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.

Macromolecular conjugates of transport inhibitors: new tools for probing topography of anion transport proteins

Macromolecular-conjugated, water-soluble, membrane-impermeant compounds were designed and assessed as topological probes for chloride-transporting agencies. The novel compounds were derivatives of either disulfonic stilbene (DS) and benzylaminoethylsulfonate (BS), "classical" inhibitors of erythrocyte chloride-bicarbonate exchange, or of phenylanthranilates (PA), high-affinity blockers of epithelial chloride channels. Covalent reactive derivatives of various DS, BS, and PA were synthesized and coupled either directly to polyethylene glycol or via spacer arms of different lengths to dextrans. The macromolecular conjugates were demonstrably inhibitory to red blood cell anion exchange when the ligands were appropriately coupled: inhibitory efficacy strongly depended on the chemical structure of the coupled ligand and the spacer length between the inhibitory moiety and the macromolecule. Mechanistic studies indicated that impermeant DS and PA derivatives acted exofacially on sites, which although different in their affinity for chloride, shared geographical proximity. BS derivatives were unique in that they affected transport from either surface. The results suggest asymmetric aqueous access routes leading to the functional domain of the anion transporter from either membrane surface.

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.

N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) as a photoaffinity probe for identifying membrane components containing the modifier site of the human red blood cell anion exchange system.

Exposure of cells to intense light with the photoactivatable reagent, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), present in the external medium results in irreversible inhibition of chloride or sulfate exchange. This irreversible inhibition seems to result from covalent reaction with the same sites to which NAP-taurine binds reversibly in the dark. As shown in the preceding paper, high chloride concentrations decrease the reversible inhibition by NAP-taurine in the dark, in a manner suggesting that NAP-taurine and chloride compete for the modifier site of the anion transport system. In a similar fashion, high chloride concentrations in the medium during exposure to light cause a decrease in both the irreversible binding of NAP-taurine to the membrane and the inhibition of chloride exchange. Most of the chloride-sensitive irreversibly bound NAP-taurine is found in the 95,000 dalton polypeptide known as band 3 and, after pronase treatment of intact cells, in the 65,000 dalton fragment of this protein produced by proteolytic cleavage. After chymotrypsin treatment of ghosts, the NAP-taurine is localized in the 17,000 dalton transmembrane portion of this fragment. Although the possible involvement of minor labeled proteins cannot be rigorously excluded, the modifier site labeled by external NAP-taurine appears, therefore, to be located in the same portion of the 95,000 dalton polypeptide as is the transport site.

Carbonic anhydrase: chemistry, physiology, and inhibition.

Carbonic anhydrase: chemistry, physiology, and inhibition.