Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

Physical and functional links between anion exchanger-1 and sodium pump.

Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in α-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the ß1 subunit of Na(+),K(+)-ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and ß1 colocalized in renal α-intercalated cells and coimmunoprecipitated (together with the catalytic α1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and ß1 interact directly, with a Kd value of 0.81 µM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for ß1 binding. siRNA-induced knockdown of ß1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of ß1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of ß1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.

Transport of H2S and HS(-) across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl(-)/HS(-) exchange.

The rates of H2S and HS(-) transport across the human erythrocyte membrane were estimated by measuring rates of dissipation of pH gradients in media containing 250 µM H2S/HS(-). Net acid efflux is caused by H2S/HS(-) acting analogously to CO2/HCO3(-) in the Jacobs-Stewart cycle. The steps are as follows: 1) H2S efflux through the lipid bilayer and/or a gas channel, 2) extracellular H2S deprotonation, 3) HS(-) influx in exchange for Cl(-), catalyzed by the anion exchange protein AE1, and 4) intracellular HS(-) protonation. Net acid transport by the Cl(-)/HS(-)/H2S cycle is more efficient than by the Cl(-)/HCO3(-)/CO2 cycle because of the rapid H2S-HS(-) interconversion in cells and medium. The rates of acid transport were analyzed by solving the mass flow equations for the cycle to produce estimates of the HS(-) and H2S transport rates. The data indicate that HS(-) is a very good substrate for AE1; the Cl(-)/HS(-) exchange rate is about one-third as rapid as Cl(-)/HCO3(-) exchange. The H2S permeability coefficient must also be high (>10(-2) cm/s, half time <0.003 s) to account for the pH equilibration data. The results imply that H2S and HS(-) enter erythrocytes very rapidly in the microcirculation of H2S-producing tissues, thereby acting as a sink for H2S and lowering the local extracellular concentration, and the fact that HS(-) is a substrate for a Cl(-)/HCO3(-) exchanger indicates that some effects of exogenous H2S/HS(-) may not result from a regulatory role of H2S but, rather, from net acid flux by H2S and HS(-) transport in a Jacobs-Stewart cycle.

Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton.

Band 3 is the most abundant integral protein of the red blood cell membrane. It performs two critical biological functions: maintaining ionic homeostasis, by transporting Cl- and HCO3-ions, and providing mechanical stability to the erythroid membrane. Erythroid band 3 (AE1) is one of three anion exchangers that are encoded by separate genes. The AE1 gene is transcribed by two promoters: the upstream promoter produces erythroid band 3, whereas the downstream promoter initiates transcription of the band 3 isoform in kidney. To assess the biological consequences of band 3 deficiency, we have selectively inactivated erythroid but not kidney band 3 by gene targeting in mice. Although no death in utero occurred, the majority of homozygous mice die within two weeks after birth. The erythroid band 3 null mice show retarded growth, spherocytic red blood cell morphology and severe haemolytic anaemia. Remarkably, the band 3-/- red blood cells assembled normal membrane skeleton thus challenging the notion that the presence of band 3 is required for the stable biogenesis of membrane skeleton. The availability of band 3-/- mice offers a unique opportunity to investigate the role of erythroid band 3 in the regulation of membrane-skeletal interactions, anion transport and the invasion and growth of malaria parasite into red blood cells.

Anion exchanger 1 interacts with nephrin in podocytes.

The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.

[Correlation of band 3 protein in erythrocytes with physiological status of middle-aged and elderly people].

OBJECTIVE: To analyze the correlation of band 3 protein of erythrocytes with physiological conditions of middle-aged and elderly people, and to investigate the expression of band 3 protein under different physiological status. METHODS: Cluster randomized sampling method for selecting subjects . Physical examination data and blood samples of 218 middle-aged and elderly people (80 males and 138 females) were collected. SDS-polyacrylamide gel electrophoresis was used to analyze band 3 protein in erythrocyte membrane. The difference of band 3 protein in different groups was analyzed by T Test and One-Way ANOVA. Correlation and regression of band 3 protein with physiological status was analyzed by Pearson correlation and linear regression. RESULTS: There was a significantly positive correlation (r =0.149) between the level of band 3 protein and physical activity (P <0.05), and a negative correlation (r = -0.156) between the level of band 3 protein and systolic blood pressure (P <0.05). The correlation of band 3 protein with gender, age, BMI and diastolic blood pressure (P >0.05) is not significant. The band 3 protein level of workers with medium activity was 24. 09% , which was significantly higher than that of light activity workers (23.42%) (P <0.05). The subjects with hypertension were proved to have a significantly lower level of band 3 protein (23.33%) than normal individuals (24.20%) (P < 0.05). CONCLUSION: The level of band 3 protein was positively correlated with physical activity and negatively correlated with systolic blood pressure.

Hydrogen ion dynamics in human red blood cells.

Our understanding of pH regulation within red blood cells (RBCs) has been inferred mainly from indirect experiments rather than from in situ measurements of intracellular pH (pH(i)). The present work shows that carboxy-SNARF-1, a pH fluorophore, when used with confocal imaging or flow cytometry, reliably reports pH(i) in individual, human RBCs, provided intracellular fluorescence is calibrated using a 'null-point' procedure. Mean pH(i) was 7.25 in CO(2)/HCO(3)(-)-buffered medium and 7.15 in Hepes-buffered medium, and varied linearly with extracellular pH (slope of 0.77). Intrinsic (non-CO(2)/HCO(3)(-)-dependent) buffering power, estimated in the intact cell (85 mmol (l cell)(-1) (pH unit)(-1) at resting pH(i)), was somewhat higher than previous estimates from cell lysates (50-70 mmol (l cell)(-1) (pH unit)(-1)). Acute displacement of pH(i) (superfusion of weak acids/bases) triggered rapid pH(i) recovery. This was mediated via membrane Cl(-)/HCO(3)(-) exchange (the AE1 gene product), irrespective of whether recovery was from an intracellular acid or base load, and with no evident contribution from other transporters such as Na(+)/H(+) exchange. H(+)-equivalent flux through AE1 was a linear function of [H(+)](i) and reversed at resting pH(i), indicating that its activity is not allosterically regulated by pH(i), in contrast to other AE isoforms. By simultaneously monitoring pH(i) and markers of cell volume, a functional link between membrane ion transport, volume and pH(i) was demonstrated. RBC pH(i) is therefore tightly regulated via AE1 activity, but modulated during changes of cell volume. A comparable volume-pH(i) link may also be important in other cell types expressing anion exchangers. Direct measurement of pH(i) should be useful in future investigations of RBC physiology and pathology.

Adaptation of intercalated cells along the collecting duct to systemic acid/base changes.

Collecting duct intercalated cells respond to short-term acid/base perturbations by rapidly shuttling H(+)-ATPase to and from the plasma membrane. Purkerson et al. provide information on the regulation of the anion transporters during chronic acidosis and acute recovery (alkalosis). They found that the major mechanism for both acute and chronic states is regulation of both the H(+)-ATPase and the anion exchangers plus changes in the overall expression level of these anion transporters in chronic adaptation.

Adaptation to metabolic acidosis and its recovery are associated with changes in anion exchanger distribution and expression in the cortical collecting duct.

It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in ß-intercalated cells and the basolateral anion exchanger (AE1) in α-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and α-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6-20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base.

Anion conductance of the human red cell is carried by a maxi-anion channel.

Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance.

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation.

Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D.

Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.

Familial renal tubular acidosis.

The kidney maintains systemic acid-base homeostasis through proximal tubular reclamation of filtered bicarbonate, and excretion of the daily mineral acid load by collecting duct type A intercalated cells. Impairment of either process produces renal tubular acidosis (RTA). This article will provide an overview of familial forms of proximal and distal renal tubular acidosis (pRTA and dRTA). Recessive pRTA with ocular and central nervous system abnormalities is caused by loss-of-function mutations in basolateral membrane Na-HCO3- cotransporter NBCe1/ SLC4A4. Recessive dRTA with deafness is caused by loss-of-function mutations in either of 2 subunits of the vacuolar H+-ATPase (V-ATPase) of intercalated cells; the B1 subunit of the V1 cytoplasmic ATPase complex, and the a4 subunit of the V0 transmembrane pore complex. Dominant and recessive forms of dRTA are also caused by loss-of-function mutations in the basolateral membrane AE1 Cl-/HCO3- exchanger of the type A intercalated cell. The dominant AE1 dRTA mutations are accompanied by mild or asymptomatic erythroid changes, while the erythroid dyscrasias accompanying recessive AE1 dRTA mutations can be mild or severe. Recessive mixed proximal-distal RTA is caused by loss-of-function mutations of the cytoplasmic carbonic anhydrase II. Hyperkalemic RTA accompanied by hypertension (pseudohypoaldosteronism type 2 [PHA2]) is caused by dominant gain-of-function mutations in the kinases WNK1 and WNK4. Hyperkalemic RTA accompanied by volume depletion is caused by loss-of-function mutations in genes encoding the mineralocorticoid receptor or the epithelial Na+ channel (ENaC) subunits. Additional RTA genes identified in knockout mice may lead to identification of additional human RTA genes.

Saccharomyces cerevisiae: First Steps to a Suitable Model System To Study the Function and Intracellular Transport of Human Kidney Anion Exchanger 1.

Saccharomyces cerevisiae has been frequently used to study biogenesis, functionality, and intracellular transport of various renal proteins, including ion channels, solute transporters, and aquaporins. Specific mutations in genes encoding most of these renal proteins affect kidney function in such a way that various disease phenotypes ultimately occur. In this context, human kidney anion exchanger 1 (kAE1) represents an important bicarbonate/chloride exchanger which maintains the acid-base homeostasis in the human body. Malfunctions in kAE1 lead to a pathological phenotype known as distal renal tubular acidosis (dRTA). Here, we evaluated the potential of baker's yeast as a model system to investigate different cellular aspects of kAE1 physiology. For the first time, we successfully expressed yeast codon-optimized full-length versions of tagged and untagged wild-type kAE1 and demonstrated their partial localization at the yeast plasma membrane (PM). Finally, pH and chloride measurements further suggest biological activity of full-length kAE1, emphasizing the potential of S. cerevisiae as a model system for studying trafficking, activity, and/or degradation of mammalian ion channels and transporters such as kAE1 in the future.IMPORTANCE Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.

Hereditary stomatocytosis and cation-leaky red cells--recent developments.

The hereditary stomatocytoses (HSt) are a diverse group of conditions. Common features include hemolytic anemia, a red cell cation leak and morphological changes, but the severity of the condition can vary enormously. We have previously shown that one form of HSt (cryohydrocytosis), where the monovalent cation leak is increased at low temperature, results from amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1). These substitutions appear to convert band 3 from an anion exchanger into a cation channel. More recently we found that over-hydrated hereditary stomatocytosis (OHSt) results from amino acid substitutions in Rh-associated glycoprotein (RhAG), a putative gas channel protein. Both band 3 and RhAG associate in the red cell membrane to form a macrocomplex that is thought to be involved in red cell gas exchange. In this paper I will review the data that has been published so far on the molecular basis of HSt. I will mention other similar conditions that cause either a cation leak or stomatocytosis or both, and consider the mechanisms of red cell shape change and permeability.

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis.

BACKGROUND: Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. DESIGN AND METHODS: We report a novel variant of hereditary stomatocytosis due to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patient's erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events. RESULTS: The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na(+) content with decreased K(+)content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl(-), HCO(3)(-)) to being a cation pathway for Na(+) and K(+). Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient's red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events. CONCLUSIONS: Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis.

A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.

Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein.

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.

The effect of apical and basolateral lipids on the function of the band 3 anion exchange protein.

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type-specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein. We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM1 (Gal 1beta1, 3GalNAcbeta1, 4Gal-NeuAcalpha2, 3Galbeta1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4, 4'-dinitorsostilbene-2,2'-disulfonic acid (DNDS; a reversible stilbene) on 35SO4/SO4 exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM1 reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.

Evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte.

Soluble oligosaccharides derived from the surface of human erythrocytes were tested for their ability to competitively inhibit invasion of erythrocytes by Plasmodium falciparum, a malarial parasite. Invasion was most effectively inhibited by erythroglycan, a carbohydrate component of the band 3 transmembrane protein. The lactosamine chains of erythroglycan contributed much of the inhibitory activity. This indication of a primary parasite interaction site on band 3 supports a role for this protein in mediating the radical alterations of the erythrocyte cytoskeleton that accompany invasion.