ABSTRACT
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.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Cell Membrane/physiology , Saccharomyces cerevisiae , Anion Exchange Protein 1, Erythrocyte/genetics , Biological Transport , Genetic Vectors , Microorganisms, Genetically-Modified , Plasmids , Saccharomyces cerevisiae/genetics , Transformation, GeneticABSTRACT
Erythrocytes undergo programmed cell death, similar to apoptosis, known as eryptosis. This process is a result of several factors including hyperosmolarity, oxidative stress, and exposure to xenobiotics, and is characterized by the breakdown of membrane phospholipid asymmetry, the clustering of band 3, and the generation of red blood cell-derived microparticles. Under pathological conditions, the liver is the primary site of erythrocyte clearance and plays an important role in iron recycling. Phosphatidylserine exposure and band-3 clustering on eryptotic erythrocytes represent mainly pro-phagocytic signals. Further, the percentage of eryptotic erythrocytes is enhanced in the circulating blood of patients with hepatic failure, hyperbilirubinemia, and nonalcoholic steatohepatitis. In this review, we concentrate on recent progress regarding the pathophysiological roles of eryptosis in liver diseases.
Subject(s)
Eryptosis/physiology , Liver Diseases/physiopathology , Anion Exchange Protein 1, Erythrocyte/physiology , Calcium/blood , Cell-Derived Microparticles/metabolism , Ceramides/blood , Cytosol/chemistry , Erythrocyte Aging , Erythrocyte Membrane/chemistry , Humans , Iron/metabolism , Liver Diseases/blood , Membrane Lipids/blood , Phosphatidylserines/blood , Reactive Oxygen Species/bloodABSTRACT
In the renal collecting duct, intercalated cells regulate acid-base balance by effluxing protons through the v-H+-ATPase, and bicarbonate via apical pendrin or the basolateral kidney anion exchanger 1 (kAE1). Additionally, collecting duct cells play an essential role in transepithelial absorption of sodium and chloride. Expression of kAE1 in polarized MDCK I cells was previously shown to decrease trans-epithelial electrical resistance (TEER), suggesting a novel role for kAE1 in paracellular permeability. In our study, we not only confirmed that inducible expression of kAE1 in mIMCD3 cells decreased TEER but we also observed (i) increased epithelial absolute permeability to both sodium and chloride, and (ii) that this effect was dependent on kAE1 activity. Further, kAE1 regulated tight junction properties through the tight junction protein claudin-4, a protein with which it physically interacts and colocalizes. These findings unveil a novel interaction between the junctional protein claudin-4 and the kidney anion exchanger, which may be relevant to ion and/or pH homeostasis.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Claudin-4/metabolism , Kidney Tubules, Collecting/cytology , Tight Junctions/metabolism , Animals , Biological Transport , Cell Line , Cell Membrane Permeability , Chlorides/metabolism , Electric Impedance , Kidney/metabolism , Mice , Sodium/metabolismABSTRACT
AIM: To analyze the variants of the potential causative genes in five Chinese patients with primary distal renal tubular acidosis (dRTA) from five unrelated families, and to explore their possible genotype-phenotype correlations, so as to raise the awareness of the disease. METHODS: Variants were identified by next generation sequencing. Clinical features and biochemical findings at the first presentation, as well as at follow-up visits were also investigated. One hundred unrelated healthy subjects were selected to evaluate each of the novel mutations found in this study. RESULTS: A total of seven different mutations in the ATP6V0A4, ATP6V1B1, and SLC4A1 genes, the three main causative genes of dRTA, were detected in 4/5 patients. In patient I a novel heterozygous intronic mutation (c.639 + 1G>A) in the ATP6V0A4 gene was identified along with a heterozygous nonsense variant (c.580C>T, p.Arg194*). Two novel heterozygous missense mutations of the ATP6V1B1 gene (c.409C>T, p.Pro137Ser; c.904C>T, p.Arg302Trp) were identified in patient II. In patient III 2 novel heterozygous duplications (c.1504dupT, p.Tyr502Leufs*22; c.2351dupT, p.Phe785Ilefs*28) were found. Thus, these three patients all were compound heterozygotes leading to dRTA. These findings are consistent with the known autosomal recessive inheritance pattern of this disease. Furthermore, a de novo heterozygous missense mutation previously reported (c.1765C>A, p.Arg589Ser) in the SLC4A1 gene was observed in patient IV. No mutations in any of the known dRTA-related causative genes were found in the patient V. CONCLUSIONS: In the present study we identified 7 mutations, including 5 novel variants, in the three genes previously correlated with dRTA, enriching the human gene mutation database (HGMD). In addition, our lack of findings in these three genes for patient V suggests that other genes may contribute to dRTA in some cases.
Subject(s)
Acidosis, Renal Tubular/genetics , Adolescent , Adult , Anion Exchange Protein 1, Erythrocyte/genetics , Anion Exchange Protein 1, Erythrocyte/physiology , Asian People/genetics , Child , Child, Preschool , China , DNA Mutational Analysis , Exons , Female , Genetic Association Studies/methods , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Middle Aged , Mutation , Mutation, Missense , Pedigree , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/physiologyABSTRACT
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.
Subject(s)
Acidosis, Renal Tubular/enzymology , Anion Exchange Protein 1, Erythrocyte/physiology , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/enzymology , Vacuolar Proton-Translocating ATPases/physiology , Animals , Anion Exchange Protein 1, Erythrocyte/genetics , Male , Mice , Models, BiologicalABSTRACT
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.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Cell Membrane/metabolism , Kidney/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Anion Exchange Protein 1, Erythrocyte/deficiency , Anion Exchange Protein 1, Erythrocyte/genetics , Cell Line , Cell Membrane/pathology , Cells, Cultured , Homeostasis/physiology , Humans , Kidney/pathology , Kidney Tubules, Collecting/metabolism , Kidney Tubules, Collecting/pathology , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Protein Binding , RNA, Small Interfering/pharmacology , Sodium-Potassium-Exchanging ATPase/drug effectsABSTRACT
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.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Cell Membrane/metabolism , Erythrocytes/metabolism , Hydrogen Sulfide/metabolism , Biological Transport/physiology , Diffusion , HumansABSTRACT
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.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Aged , Aged, 80 and over , Blood Pressure , Electrophoresis, Polyacrylamide Gel/methods , Female , Humans , Male , Middle Aged , Motor Activity , Physiological Phenomena , Sampling Studies , Surveys and QuestionnairesSubject(s)
Acidosis, Renal Tubular , Acidosis, Renal Tubular/genetics , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/physiology , Anion Exchange Protein 1, Erythrocyte/genetics , Anion Exchange Protein 1, Erythrocyte/physiology , Fanconi Syndrome/etiology , Genes, Recessive , Humans , Lysosomes/metabolism , Mutation , Sodium-Bicarbonate Symporters/genetics , Sodium-Bicarbonate Symporters/physiology , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/physiology , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIc/physiology , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/physiologyABSTRACT
OBJECTIVES: To examine erythrocyte band III transport protein (SLC4A1), erythrocyte oxalate flux, and plasmatic, cellular, and urine oxalate concentrations and blood gas analyses in calcium oxalate monohydrate stone-forming patients (COM) in comparison with normal controls (NC). METHODS: Isolated red cells from 51 NC and 25 COM cases were divided for cellular oxalate measurement and for measurement of transcellular erythrocyte oxalate flux (pH 7.48-8.24). SLC4A1 protein levels were determined by Western blot analyses. Plasmatic and urinary oxalate levels and the venous blood gas analysis were measured simultaneously. RESULTS: SLC4A1 protein levels were significantly higher in COM (8.76 ± 2.12) than in NC (4.17 ± 0.61; P < .02). Cellular oxalate and venous HCO(3)(-) were significantly lower in COM (2.35 ± 0.26 µmol/L) and (24.06 ± 0.24 mmo/l) than in NC (4.03 ± 0.49 µmol/L; P < .05) and (24.93 ± 0.17 mmol/L; P < .01). Urinary oxalate was significantly higher in COM (0.31 ± 0.02 mmol/L) than in NC (0.25 ± 0.01 mmol/L; P < .04). The erythrocyte transmembrane oxalate flux correlated with the pH value and with the urinary oxalate in both groups (r = .25-.55; P = .01). With increased pH values, the oxalate flux showed inverse effects in both groups. CONCLUSIONS: SLC4A1 associated changes of HCO(3)(-) and pH levels influenced the cellular oxalate levels and urinary oxalate clearance. Under normal conditions (pH 7.55) the oxalate efflux in COM was comparable with the acid stimulated oxalate efflux in NC. The addition of HCO(3)(-) compensated the flux of COM stone formers to the levels of normal controls.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Calcium Oxalate , Oxalates/metabolism , Urinary Calculi/metabolism , Female , Humans , Male , Middle AgedABSTRACT
The present study investigated the changes in several erythrocyte oxidative stress biomarkers in hypoxic elderly individuals to analyze the deleterious effects of low oxyhemoglobin saturation in an elderly population. We collected blood samples from one normoxic middle-aged group and two groups composed of individuals older than 75 years of age: one normoxic group and one hypoxic group. Aging appeared to provoke a defective erythrocyte antioxidant defense associated with increased oxidative damage in the elderly population. Acute hypoxia activated an insufficient antioxidant defense response as suggested by the oxidative damage observed. The oxidative imbalance presented in older participants and increased in hypoxia participants had a direct effect on glyceraldehyde-3-phosphate dehydrogenase cell distribution. Oxidative stress levels altered Band 3 protein and mediated caspase-3 activation in erythrocyte from the aged group although it was not extended to hypoxic individuals. Therefore, aged participants appeared to activate an insufficient antioxidant response against hypoxia-related oxidative stress.
Subject(s)
Adaptation, Physiological , Erythrocytes/physiology , Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)/physiology , Hypoxia/physiopathology , Oxidative Stress , Aged , Aged, 80 and over , Anion Exchange Protein 1, Erythrocyte/physiology , Antioxidants/physiology , Caspase 3/physiology , Catalase/physiology , Erythrocytes/enzymology , Female , Glutathione Reductase/physiology , Humans , Hypoxia/enzymology , Male , Superoxide Dismutase/physiologyABSTRACT
Recent evidence has shown that plasma fibrinogen, a major cardiovascular risk factor, interacts with the erythrocyte membrane and acts to influence blood flow via erythrocyte nitric oxide (NO) modulation. In the present pioneer in-vitro study, whole blood samples were harvested from healthy subjects and aliquots were incubated in the absence (control aliquots) and presence of fibrinogen at different degrees of band 3 phosphorylation, and the levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Hyperfibrinogenemia interferes with erythrocyte NO mobilization without changing its efflux in a way that seems to be dependent of the degree of band 3 phosphorylation. In presence of higher fibrinogen concentrations the NO efflux is reinforced when band 3 is phosphorylated (p < 0.001). Higher levels of nitrite, nitrate and GSNO were documented (p < 0.05). However, the mechanisms by which fibrinogen signalling modulates erythrocyte function remain to be clarified and are currently under study. These conditions may be considered an approach to be followed in blood storage for transfusions.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Erythrocytes/metabolism , Fibrinogen/physiology , Nitric Oxide/blood , Protein Processing, Post-Translational , Anion Exchange Protein 1, Erythrocyte/chemistry , Biological Transport/drug effects , Biological Transport/physiology , Diffusion , Dipeptides/pharmacology , Erythrocyte Membrane/metabolism , Erythrocytes/drug effects , Fibrinogen/analysis , Flavonoids/pharmacology , Genistein/analogs & derivatives , Glutathione/analogs & derivatives , Glutathione/pharmacology , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Male , Nitrates/blood , Nitrites/blood , Nitro Compounds/pharmacology , Osmolar Concentration , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Syk KinaseABSTRACT
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.
Subject(s)
Acidosis, Renal Tubular/genetics , Animals , Anion Exchange Protein 1, Erythrocyte/genetics , Anion Exchange Protein 1, Erythrocyte/physiology , Disease Models, Animal , Humans , Hypercalciuria/etiology , Hypokalemia/etiology , Mice , Mutation , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
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.
Subject(s)
Acid-Base Equilibrium/physiology , Adaptation, Physiological/physiology , Kidney Tubules, Collecting/physiology , Proton-Translocating ATPases/physiology , Acidosis/physiopathology , Alkalosis/physiopathology , Animals , Anion Exchange Protein 1, Erythrocyte/physiology , Anion Transport Proteins/physiology , Kidney Tubules, Collecting/cytology , Membrane Transport Proteins/physiology , Models, Animal , RabbitsABSTRACT
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.
Subject(s)
Erythrocytes/physiology , Protons , Anion Exchange Protein 1, Erythrocyte/physiology , Benzopyrans/chemistry , Benzopyrans/metabolism , Bicarbonates , Buffers , Carbon Dioxide , Chlorides/metabolism , Fluorescence , Humans , Hydrogen-Ion Concentration , Naphthols/chemistry , Naphthols/metabolism , Rhodamines/chemistry , Rhodamines/metabolismABSTRACT
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.
Subject(s)
Acidosis, Renal Tubular/genetics , Anemia, Hemolytic, Congenital/genetics , Anion Exchange Protein 1, Erythrocyte/genetics , Mutation, Missense , Point Mutation , Acidosis, Renal Tubular/complications , Amino Acid Substitution , Anemia, Hemolytic, Congenital/complications , Anemia, Macrocytic/complications , Anemia, Macrocytic/drug therapy , Anion Exchange Protein 1, Erythrocyte/physiology , Child , Codon/genetics , Consanguinity , Ethnicity/genetics , Homozygote , Humans , India/epidemiology , Infant , Introns/genetics , Pedigree , Polymorphism, Restriction Fragment Length , Tuberculosis, Multidrug-Resistant/complicationsABSTRACT
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.
Subject(s)
Acidosis/physiopathology , Adaptation, Physiological/physiology , Alkalosis/physiopathology , Anion Transport Proteins/physiology , Kidney Tubules, Collecting/physiology , Acid-Base Equilibrium/physiology , Alkalosis/chemically induced , Animals , Anion Exchange Protein 1, Erythrocyte/physiology , Disease Models, Animal , Female , Kidney Tubules, Collecting/pathology , Membrane Transport Proteins/physiology , Proton-Translocating ATPases/physiology , Rabbits , Sodium Bicarbonate/administration & dosageABSTRACT
Adducin is an alpha, beta heterotetramer that performs multiple important functions in the human erythrocyte membrane. First, adducin forms a bridge that connects the spectrin-actin junctional complex to band 3, the major membrane-spanning protein in the bilayer. Rupture of this bridge leads to membrane instability and spontaneous fragmentation. Second, adducin caps the fast growing (barbed) end of actin filaments, preventing the tetradecameric protofilaments from elongating into macroscopic F-actin microfilaments. Third, adducin stabilizes the association between actin and spectrin, assuring that the junctional complex remains intact during the mechanical distortions experienced by the circulating cell. And finally, adducin responds to stimuli that may be important in regulating the global properties of the cell, possibly including cation transport, cell morphology and membrane deformability. The text below summarizes the structural properties of adducin, its multiple functions in erythrocytes, and the consequences of engineered deletions of each of adducin subunits in transgenic mice.
Subject(s)
Calmodulin-Binding Proteins/blood , Erythrocyte Membrane/physiology , Erythrocytes/physiology , Actins/blood , Animals , Anion Exchange Protein 1, Erythrocyte/physiology , Blood Proteins , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/physiology , Erythrocyte Deformability/physiology , Erythrocyte Membrane/ultrastructure , Glucose Transport Proteins, Facilitative/blood , Homeostasis , Humans , Lipid Bilayers , Membrane Proteins/blood , Mice , Mice, Transgenic , Spectrin/metabolismABSTRACT
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.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Membrane Proteins/physiology , Podocytes/metabolism , Adult , Albuminuria/metabolism , Amino Acid Sequence , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Cells, Cultured , Female , Fluorescent Antibody Technique , Humans , Kidney Glomerulus/pathology , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Protein Serine-Threonine Kinases/physiology , Two-Hybrid System Techniques , XenopusABSTRACT
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.