ABSTRACT
In this study we examined the impact of 3-day hypoxia of varying degrees on the viability, proliferative and secretory activity of endothelial cells in human umbilical vein (HUVEC). The gas mixture of the three components (%) was used: 1) 10 O2, 5 CO2, 85 Ar; 2) 5 O2, 5 CO2, 90 Ar and 3) 1 O2, 5 CO2, 94 Ar. The HUVEC, cultivated in CO2-incubator under conditions of atmospheric oxygen (21% O2) were the controls. Comprehensive assessment of the results after has shown that 3-day HUVEC cultivating in the presence of 1% O2 led to pathological activation of endotheliocytes: increased NO synthesis combined with the marked secretion of endothelin-1, IL-6, IL-8 and TNF-alpha, sVCAM-1, sE-cadherin and of sE-selectin, VEGF-A and bFGF, and slow proliferation. When HUVEC were cultivated at 10% O2 and 5% O2, the level of basal secretion of the substances listed above was the least against the background of increased proliferative activity. The results showing the changes in the secretory activity of endothelial cells when cultivated under the conditions of atmospheric oxygen levels have demonstrate HUVEC activation, because the secretion of NO, IL-6, IL-8 and von Willebrand factor after 3 days of cultivation in 21% 02 exceeded that in the case of 10 and 5% O2. Thus, a gaseous medium with reduced oxygen content of up to 5% provides more physiological conditions for HUVEC cultivation. The maximum proliferative activity of HUVEC with minimal basal secretion proved such a composition to be comfortable. Increasing the oxygen content to the atmospheric level leads to the activation of endotheliocytes with signs of endothelial dysfunction, and the critical reduction in oxygen to 1% causes the development of endothelial dysfunction and reduces the proliferative potential.
Subject(s)
Gene Expression/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Oxygen/pharmacology , Cadherins/genetics , Cadherins/metabolism , Cell Hypoxia , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , E-Selectin/genetics , E-Selectin/metabolism , Endothelin-1/genetics , Endothelin-1/metabolism , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Nitric Oxide/agonists , Nitric Oxide/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , von Willebrand Factor/genetics , von Willebrand Factor/metabolismABSTRACT
Hypercytokinemia plays a key role in the pathogenesis of systemic inflammatory response syndrome (SIRS). Monocytes are the main source of cytokines in the early inflammatory phase. Simultaneous stimulation of toll-like receptors (TLRs) and triggering receptor expressed on myeloid cells (TREM-1) activating receptor on monocytes results in the amplification of the inflammatory signal and multiple increase in proinflammatory cytokine production. The dynamics of those receptors expression on monocyte surface of patients with uncomplicated SIRS course followed coronary artery bypass surgery (CABG) was studied. The increase in TLR2 and TREM-1 expression on the first day after CABG induces proinflammatory and amplification potentials of monocytes in that period. The decrease in TLR2 surface expression on the seventh day compared to the preoperative values can be regarded as a mechanism limiting inflammatory response. The highest level of TLR2, TLR4, and TREM-1 surface expression was observed in CD14(hi)CD16(+) monocyte subpopulation, confirming its proinflammatory profile.
ABSTRACT
We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).
Subject(s)
Anion Transport Proteins , Antiporters , Chloride-Bicarbonate Antiporters/metabolism , Conserved Sequence/physiology , Cytoplasm/metabolism , Ion Transport/physiology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Acids/pharmacokinetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Biological Transport, Active/physiology , Chloride-Bicarbonate Antiporters/chemistry , Female , Hydrogen-Ion Concentration , Kinetics , Mutagenesis, Site-Directed , Mutation, Missense , Protein Structure, Tertiary/physiology , SLC4A Proteins , Structure-Activity Relationship , XenopusABSTRACT
In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte Na(+)/H(+) exchange activity (xoNHE) by hypertonic shrinkage required Cl(-), with an EC(50) for bath [Cl(-)] of approximately 3mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by Cl(-) transport inhibitors. Chelation of intracellular Ca(2+) by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of Ca(2+)-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl(-)-dependent increase in c-Jun NH(2)-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of Na(+)/H(+) exchange in Xenopus oocytes requires Cl(-) and is mediated by activation of JNK.
Subject(s)
Chlorides/metabolism , Mitogen-Activated Protein Kinases/metabolism , Sodium-Hydrogen Exchangers/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Anions/pharmacology , Anthracenes/pharmacology , Calcium/metabolism , Chelating Agents/pharmacology , Diuretics/pharmacology , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Female , Hydrogen/metabolism , Hydrogen-Ion Concentration , Hypertonic Solutions/chemistry , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oocytes/physiology , Osmolar Concentration , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium/metabolism , Sodium Radioisotopes/metabolism , Xenopus laevisABSTRACT
The role of intracellular pH (pH(i)) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated (36)Cl(-) efflux from Xenopus oocytes during imposed variation of extracellular pH (pH(o)) or variation of pH(i) at constant pH(o). Wild-type AE2-mediated (36)Cl(-) efflux displayed a steep pH(o) vs. activity curve, with pH(o(50)) = 6.91 +/- 0.04. Sequential NH(2)-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pH(o(50)) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pH(i) at constant pH(o) and were shown to be neither substrates nor inhibitors of AE2-mediated Cl(-) transport. At constant pH(o), AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH(2)-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.
Subject(s)
Anion Transport Proteins , Antiporters , Hydrogen-Ion Concentration , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Acids/pharmacology , Animals , Bicarbonates/pharmacokinetics , Butyrates/pharmacokinetics , Carbon Radioisotopes , Chlorides/pharmacokinetics , Cytoplasm/metabolism , Membrane Proteins/genetics , Microelectrodes , Mutagenesis/physiology , Oocytes/physiology , Protein Structure, Tertiary , Protons , Radioisotopes , SLC4A Proteins , XenopusABSTRACT
K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.
Subject(s)
Chlorides/metabolism , Potassium/metabolism , Symporters/chemistry , Animals , Cytoplasm/chemistry , Humans , Mice , Mutation , Rats , Structure-Activity Relationship , Symporters/genetics , Symporters/physiology , K Cl- CotransportersABSTRACT
The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.
Subject(s)
Calcium Channels/metabolism , Peptide Fragments/pharmacology , Proteins/chemistry , Amino Acid Sequence , Animals , Calcium Channels/physiology , Molecular Sequence Data , Patch-Clamp Techniques , Peptide Fragments/chemistry , Permeability , TRPP Cation Channels , XenopusABSTRACT
Among human bicarbonate transporters, two major gene families encode Na-independent Cl(-)/HCO(3)(-) exchangers: the SLC4 anion exchanger (AE) family, and the SLC26 "sulfate permease" anion transporter family. The SLC4 AE family contains at least three genes, and comprises a subfamily within the larger and phylogenetically more ancient bicarbonate transporter superfamily that includes the Na bicarbonate cotransporters (NBC) and the Na-driven Cl/base exchangers. Mutations in the human AE1 gene cause autosomal dominant spherocytic anemia and distal renal tubular acidosis of both dominant and recessive forms. Anemia is also associated with AE1 mutations in mouse, cow, and zebrafish. Naturally occurring mutations in the human AE2 and AE3 genes have not been detected. The SLC26 family in humans consists of at least 10 members, and includes anion exchangers which exchange chloride for bicarbonate, hydroxyl, sulfate, formate, iodide, and/or oxalate. Mutations in three of these genes cause hereditary disease, including chondrodysplasia (SLC26A2, DTD), diarrhea (A3, down-regulated in adenoma/chloride-losing diarrhea protein: DRA/CLD), and goiter/deafness syndrome (A4, pendrin). Little is known about the acute regulation of these modulators of intracellular and compartmental pH and volume.
Subject(s)
Chloride-Bicarbonate Antiporters/physiology , Sodium-Bicarbonate Symporters/physiology , Animals , Biological Transport/physiology , Humans , Hydrogen-Ion Concentration , Sulfates/metabolismABSTRACT
The distribution of the binding sites for kaliotoxin (KTX), a blocker of voltage-dependent K(+) channels, was studied with quantitative autoradiography in adult rat brain and during postnatal brain maturation. Iodinated KTX bound specifically to tissue sections with a high affinity (K(d) = 82 pM) and a maximal binding capacity of 13.4 fmol/mg protein. The distribution of KTX binding sites within the central nervous system was heterogeneous. The highest densities were found in the neocortex, hypothalamus, dentate gyrus, bed nucleus of the stria terminalis, and parabrachial nuclei. The lowest level was observed in the white matter. From postnatal day 5 onward, KTX binding sites were detectable only in the hindbrain. The density of KTX binding sites in whole brain drastically increased after postnatal day 15 to achieve adult levels at postnatal day 60 in the whole brain. Bath application of KTX to Xenopus laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels potently and Kv1.2 channels less potently, with respective K(d) values of 0.1, 1.5, and 25 nM. KTX affinities for each of these channels expressed in mammalian cells were about 10-fold lower. A comparison of the distribution of KTX binding sites with that of Kv1 channel polypeptides, together with the pharmacology of KTX block, suggests that the principal targets for KTX in rat brain are K(+) channels containing Kv1.1 and Kv1.3 alpha-subunits.
Subject(s)
Brain Chemistry/physiology , Brain/anatomy & histology , Potassium Channel Blockers , Potassium Channels, Voltage-Gated , Potassium Channels , Scorpion Venoms/pharmacology , Aging/metabolism , Animals , Autoradiography , Binding Sites/drug effects , Brain/growth & development , Cell Line , Electric Stimulation , Electrophysiology , Humans , Kidney/metabolism , Kv1.1 Potassium Channel , Kv1.2 Potassium Channel , Kv1.3 Potassium Channel , Male , Mice , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Xenopus laevisABSTRACT
Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.
Subject(s)
Anemia, Sickle Cell/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosome Mapping , Symporters , Thalassemia/metabolism , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Amino Acid Sequence , Anemia, Sickle Cell/pathology , Animals , Antibody Specificity , Base Sequence , Biological Transport/genetics , Carrier Proteins/immunology , Chlorides/pharmacokinetics , Cloning, Molecular , Cross Reactions , DNA, Complementary , Erythrocytes/chemistry , Erythrocytes/cytology , Erythrocytes/metabolism , Gene Expression Regulation/physiology , Glycosylation , Humans , Kidney/cytology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oligonucleotide Probes , Oocytes/physiology , Potassium/pharmacokinetics , Precipitin Tests , Protein Biosynthesis/physiology , RNA, Messenger/analysis , Rabbits , Rats , Thalassemia/pathology , Transfection , Xenopus , K Cl- CotransportersABSTRACT
Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.
Subject(s)
Anion Transport Proteins , Anti-Bacterial Agents/pharmacology , Antiporters/antagonists & inhibitors , Fluorescent Dyes/pharmacology , Isoxazoles/pharmacology , Membrane Proteins/antagonists & inhibitors , Polyamines/pharmacology , Sharks/metabolism , Animals , Barbiturates/pharmacology , Chloride-Bicarbonate Antiporters , Cholestanols/pharmacology , Dihydropyridines/pharmacology , Dose-Response Relationship, Drug , Erythrocytes , HL-60 Cells , Humans , Inhibitory Concentration 50 , Kinetics , Mice , Oocytes/metabolism , SLC4A Proteins , Xenopus/embryologyABSTRACT
We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/metabolism , Chlorides/metabolism , Animals , Anion Exchange Protein 1, Erythrocyte/chemistry , Anion Exchange Protein 1, Erythrocyte/genetics , Cell Line , Cell Membrane/metabolism , Chlorides/pharmacokinetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Mice , Microinjections , Mutagenesis, Site-Directed , Oocytes/metabolism , Protein Biosynthesis/genetics , RNA, Complementary , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Transfection/genetics , XenopusABSTRACT
In the course of experiments to define regulation by intracellular pH (pHi) of the AE2 anion exchanger expressed in Xenopus oocytes, we discovered an unexpected regulation of AE2 by NH4+. Intracellular acidification produced by extracellular acidification or produced by equimolar substitution of NaCl with sodium acetate each inhibited AE2 activity. In contrast, intracellular acidification by equimolar substitution of NaCl with NH4Cl activated AE2-associated, trans-anion-dependent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- influx and efflux. Regulation by NH4+ was isoform specific, since neither erythroid nor kidney AE1 was activated. AE2 activation was maximal at <5 mM NH4Cl; was not mimicked by extracellular KCl, chloroquine, or polyamines; and was insensitive to amiloride, bumetanide, barium, and gadolinium. Whether NH4Cl acts directly on AE2 or on another target remains to be determined. Activation of AE2 by NH4+ may serve to sustain Cl-/HCO3- exchange activity in the presence of acidic pH in renal medulla, colon, abscesses, and other AE2-expressing acidic locales exposed to elevated NH4+ concentration.
Subject(s)
Acids/metabolism , Ammonium Chloride/pharmacology , Anion Transport Proteins , Antiporters , Membrane Proteins/metabolism , Oocytes/metabolism , Xenopus/metabolism , Alkalies/metabolism , Animals , Electrophysiology , Female , Hydrogen-Ion Concentration , Isomerism , Mice , Oocytes/cytology , Oocytes/physiology , Organelles/metabolism , Polyamines/pharmacology , SLC4A Proteins , Sodium Acetate/pharmacology , Sodium-Hydrogen Exchangers/metabolismABSTRACT
Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4(2-) cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl- influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4(2-). E699Q-mediated Cl- influx was activated by elevation of intracellular SO4(2-), but E699Q-mediated Cl- efflux was undetectable. The DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) sensitivity of E699Q-mediated SO4(2-) efflux was indistinguishable from that of wt AE1-mediated Cl- efflux. The extracellular anion selectivity of E699Q-mediated SO4(2-) efflux was similar to that of wt AE1-mediated Cl- efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl- with intracellular SO4(2-) was 1:1. Whereas SO4(2-) injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4(2-), but not of Cl- or gluconate, into oocytes expression E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl- with isethionate caused a 28-mV hyperpolarization in SO4(2-)-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl(-)-dependent depolarization of SO4(2-)-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl- was of magnitude sufficient to account for measured 35SO4(2-) efflux. Thus, AE1 E699Q-mediated SO4(2-)/Cl- exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4(2-)/Cl- exchange stoichiometry.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/metabolism , Antiporters/metabolism , Oocytes/metabolism , Sulfates/metabolism , Animals , Anion Exchange Protein 1, Erythrocyte/genetics , Bicarbonates/metabolism , Cell Line , Chloride-Bicarbonate Antiporters , Chlorides/metabolism , Female , Humans , Membrane Potentials/physiology , Mice , Mutation , Precipitin Tests , RNA, Complementary/metabolism , Solutions , Stilbenes/metabolism , Sulfur Radioisotopes , Xenopus laevisABSTRACT
Xenopus oocytes lack volume regulation and Cl/anion-exchange (AE) activity but express endogenous Na+/H+ exchange (NHE). We postulated that expression in oocytes of heterologous anion exchangers might allow regulatory volume increase (RVI) via functional coupling with endogenous NHE. Expression of neither erythroid nor kidney isoforms of AE1 conferred any form of RVI. In contrast, although AE2 expression did not confer primary RVI, it did confer on oocytes secondary RVI, with a requirement for hypotonic swelling before hypertonic shrinkage. This secondary RVI required extracellular Cl- and Na+, was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and amiloride, was bumetanide insensitive, and was blocked by prevention of intracellular alkalinization, all properties consistent with functional coupling of AE2-mediated Cl-/HCO3- exchange and endogenous NHE. RVI was unaffected by CO2-HCO3- or by partial oocyte Cl- depletion and was unrelated to the rate of oocyte shrinkage. Prior hypotonic swelling did not significantly alter subsequent hypertonic stimulation of AE2-mediated 36Cl influx or efflux. We conclude that heterologous AE2 expression suffices to confer volume regulation on Xenopus oocytes that lack intrinsic volume-regulatory mechanisms.
Subject(s)
Anion Transport Proteins , Membrane Proteins/metabolism , Oocytes/cytology , Oocytes/metabolism , Alkalies/metabolism , Animals , Antiporters/metabolism , Bicarbonates/pharmacology , Chlorides/metabolism , Extracellular Space/metabolism , Female , Hypertonic Solutions/metabolism , Hypertonic Solutions/pharmacology , Intracellular Membranes/metabolism , Isomerism , Membrane Proteins/drug effects , Membrane Proteins/physiology , SLC4A Proteins , Sodium-Hydrogen Exchangers/metabolism , Sodium-Hydrogen Exchangers/physiology , XenopusABSTRACT
The AE2 anion exchanger in pig and rabbit gastric mucosal membranes was subjected to limited proteolysis with trypsin, chymotrypsin, and papain, and to enzymatic N-deglycosylation. A monoclonal antibody to the AE2 C-terminal peptide was raised, characterized, and used to purify pig AE2 and its C-terminal cleavage products. Five distinct proteolytic cleavage sites within the AE2 transmembrane domain were defined by amino acid sequencing. The amino acid sequence of pig AE2 in the region encompassing the N-glycosylated Z-loop was also determined by RT-PCR. Tryptic cleavage of pig AE2 in the Z-loop produced C-terminal glycopeptides and was unaffected by deglycosylation, whereas the smaller rabbit AE2 C-terminal tryptic peptide lacked oligosaccharide, consistent with the respective amino acid sequences. The third consensus N-glycosylation site in pig Z-loop was heterogeneously glycosylated. Rapid papain cleavage in the Z-loop and slower cleavage in loop 7-8 produced C-terminal peptide products which were not N-glycosylated. Chymotryptic cleavage of the rabbit AE2 Z-loop required prior deglycosylation. Chymotryptic cleavage in the pig AE2 Z-loop produced C-terminal glycopeptides. Prior deglycosylation of pig AE2 unmasked novel, ionic strength-sensitive chymotryptic cleavage sites in the adjacent exofacial loop 7-8. These results provide experimental confirmation for some aspects of AE2 topography previously predicted from primary structure alone.
Subject(s)
Anion Transport Proteins , Antiporters , Gastric Mucosa/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Anions , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Chymotrypsin/metabolism , DNA, Complementary , Glycosylation , Hydrolysis , Membrane Proteins/genetics , Membrane Proteins/immunology , Molecular Sequence Data , Osmolar Concentration , Rabbits , SLC4A Proteins , Sequence Homology, Amino Acid , SwineABSTRACT
We have compared regulation by pH of AE1 (band 3)- and AE2-mediated 36Cl- uptake into Xenopus oocytes. 36Cl- influx was assayed at varying extracellular pH (pHo) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pHi) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pHo 7.0 and 63% of maximal activity at pHo 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pHo9.0 and full suppression at pHo 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2cyto/AE1memb polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1cyto/AE2memb polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 +/- 0.04 pHo units. Moreover, whereas the pH versus activity curves of AE2 Delta99 and WT AE2 were indistinguishable, AE2 Delta510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 +/- 0.13 pHo units (indistinguishable from that of AE1cyto/AE2memb). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pHi accounted for some but not all of the measured pH dependence of AE2.
Subject(s)
Anion Transport Proteins , Antiporters , Chlorides/metabolism , Membrane Proteins/metabolism , Oocytes/metabolism , Acetates/metabolism , Animals , Base Sequence , Biological Transport , DNA Primers , DNA, Complementary , Female , Homeostasis , Hydrogen-Ion Concentration , Kinetics , Membrane Proteins/biosynthesis , Mice , Molecular Sequence Data , Mutagenesis , RNA, Complementary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , SLC4A Proteins , Sequence Deletion , Time Factors , Xenopus laevisABSTRACT
Mutations in the AE1 (band 3) anion exchanger of human erythrocytes have been associated with altered red cell shape and heritable disease. The Southeast Asian Ovalocytosis (SAO) AE1 mutation, a 27 nt deletion producing the delta 400-408 form of AE1, and the AE1 Prague mutation, a 10 nt insertion producing a frameshift after AE1 aa 821 leading to premature termination, are found only in the heterozygous state. We therefore examined accumulation and function of wt AE1 polypeptide in Xenopus oocytes when coexpressed with AE1 SAO and with AE1 Prague. Our SAO construct lacked the K56E (AE1 Memphis) polymorphism present in the endogenous AE1 SAO protein. Neither mutant AE1 mediated Cl- uptake into cRNA-injected Xenopus oocytes. Coinjection of mutant and wt cRNAs led to dose-dependent inhibition of wt function by AE1 Prague, but not by SAO. Though in vitro translation of the two mutants revealed little difference in their insertion into microsomal membranes, AE1 Prague accumulated in Xenopus oocytes to lower levels than did AE1 SAO or wt. Unlike AE1 SAO polypeptide, AE1 Prague polypeptide was not detectable at the oocyte surface. Moreover, overexpression of AE1 Prague, in contrast to AE1 SAO, reduced the accumulation of wt AE1, at the oocyte surface. This inhibition occurred in the absence of detectable heteromer formation between the AE1 Prague and wt AE1 polypeptides.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/genetics , Mutation , Animals , Anion Exchange Protein 1, Erythrocyte/metabolism , Base Sequence , Biological Transport , Biopolymers , Cloning, Molecular , DNA Primers , Female , Humans , Intracellular Membranes/metabolism , Microsomes/metabolism , Molecular Sequence Data , Mutagenesis, Insertional , Oocytes , Protein Biosynthesis , RNA, Complementary , Reticulocytes , XenopusABSTRACT
Xenopus oocytes express endogenous Na+/H+ exchange activity but lack significant endogenous Cl-/HCO3- exchange activity. Coupled operation of Na+/H+ exchange and Cl-/HCO3- exchange contributes in many cell types to the cellular response to hypertonic stress. We therefore examined in Xenopus oocytes the osmotic regulation of chloride transport mediated by recombinant anion exchanger proteins AE2 and AE1. Hypotonicity was without effect on either anion transporter. Hypertonicity activated AE2-associated 36Cl- influx and efflux in a time- and osmolarity-dependent manner, whether incremental osmoles were charged or uncharged, but had no measurable effect on AE1 function. Hypertonic stimulation of AE2 was completely inhibited by Na+ removal or by addition of amiloride. In contrast, neither maneuver altered isotonic activity of AE2. Hypertonicity also induced amiloride-sensitive elevation of oocyte intracellular pH (pHi), and shifted the sigmoidal relationship of extracellular pH vs. AE2 activity > or = 0.5 units to the acid. Injection of pH 7.4 buffer into oocytes attenuated both hypertonic alkalinization and activation of AE2-associated 36Cl- influx, without inhibition of isotonic AE2 function. These data demonstrate that recombinant AE2 expressed in Xenopus oocytes is activated by increased pHi and that hypertonic activation of AE2 is secondary to hypertonic activation of Na+/H+ exchange.
Subject(s)
Alkalies/metabolism , Anion Transport Proteins , Antiporters , Hypertonic Solutions/pharmacology , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Oocytes/metabolism , Sodium-Hydrogen Exchangers/physiology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Biological Transport/drug effects , Female , Hydrogen-Ion Concentration , Hypotonic Solutions/pharmacology , Membrane Proteins/drug effects , SLC4A Proteins , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Xenopus laevisABSTRACT
cRNA encoding the murine band 3-related protein AE2 was expressed in Xenopus oocytes. AE2-mediated transport function and regulation were analyzed by unidirectional 36Cl- influx and efflux studies. AE2 cRNA-injected oocytes took up 36Cl- as much as 40-fold faster than did water-injected oocytes. AE2-mediated 36Cl- uptake increased as a function of increasing uptake time, number of days after cRNA injection, and amount of injected cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent Km for external Cl- was 5.6 mM. AE2 was defined as a Cl-/anion exchanger by two criteria: 1) 36Cl- efflux from AE2-expressing oocytes was maximally stimulated by extracellular Cl- or nitrate; AE2-associated 36Cl- efflux was supported by substitution of extracellular Cl- with other anions in the rank order bromide > isethionate > or = gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl(-)-free media containing isethionate, gluconate, or glutamate decreased subsequent AE2-associated 36Cl- uptake from Cl- media in rough proportion to the degree of intracellular Cl- depletion, whereas preincubation in nitrate medium had no effect. AE2-associated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid at half-maximally inhibitory concentrations between 0.5 and 19 microM, depending on extracellular Cl- concentration, and progressed to irreversibility at 20 degrees C with a half-time of 20-30 min. Many additional inhibitors showed lower potency for AE2 than previously reported for AE1. Although AE2 expression did not change oocyte resting intracellular pH, AE2-associated 36Cl- influx and efflux were each decreased in acid incubation medium and increased in alkaline medium.