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At least 19 recordsLinked to original sources
PubMed2000

Tissue-specific N-terminal isoforms from overlapping alternate promoters of the human AE2 anion exchanger gene.

Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b(1) corresponds to the rat variant "b" and encodes three initial residues (MTQ) in AE2b(1) isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b(2) encodes eight initial residues (MDFLLRPQ) in AE2b(2) isoform. The relative abundance of AE2b(1) and AE2b(2) mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b(1) and AE2b(2) transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.

Alternative Splicing↗
PubMed1998

Further localization of a gene for paroxysmal dystonic choreoathetosis to a 5-cM region on chromosome 2q34.

Paroxysmal dystonic choreoathetosis (PDC) is a rare neurological disorder characterized by episodes of involuntary movement, involving the extremities and face, which may occur spontaneously or be precipitated by caffeine, alcohol, anxiety, and fatigue. PDC is transmitted as an autosomal dominant trait with incomplete penetrance. A gene implicated in this paroxysmal disorder has been mapped to a 10-15 cM region on chromosome 2q31-36 in two families. We describe a third family with PDC. Two-point linkage analyses with markers linked to the candidate PDC locus were performed. A maximum two-point LOD score of 4.20 at a recombination fraction of zero was obtained for marker D2S120, confirming linkage to the distal portion of chromosome 2q. The anion exchanger gene, SLC2C, maps to this region, but the family was poorly informative for polymorphic markers within and flanking this candidate gene. Haplotype analysis revealed a critical recombination event that confines the PDC gene to a 5-cM region bounded by the markers D2S164 and D2S377. We compared the haplotype in our family with that in another chromosome 2-linked PDC family, but did not detect a region of shared genotypes. However, identifying a third family whose disease maps to the same region and narrowing the critical region will facilitate identification of the 2q-linked PDC gene.

Adult↗
PubMed2004

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes.

AE2 (SLC4A2) is the member of the Na(+)-independent anion exchanger (AE) family putatively involved in the secretion of bicarbonate to bile. In humans, three variants of AE2 mRNA have been described: the full-length transcript AE2a (expressed from the upstream promoter in most tissues), and alternative transcripts AE2b(1) and AE2b(2) (driven from alternate promoter sequences in a tissue-restricted manner, mainly in liver and kidney). These transcripts would result in AE protein isoforms with short N-terminal differences. To ascertain their translation, functionality, and membrane sorting, we constructed expression vectors encoding each AE2 isoform fused to GFP at the C-terminus. Transfected HEK293 cells showed expression of functional GFP-tagged AE2 proteins, all three isoforms displaying comparable AE activities. Primary rat hepatocytes transfected with expression vectors and repolarized in a collagen-sandwich configuration showed a microtubule-dependent apical sorting of each AE2 isoform. This shared apical sorting is liver-cell specific, as sorting of AE2 isoforms was basolateral in control experiments on polarized kidney MDCK cells. Hepatocytic apical targeting of AE2 isoforms suggests that they all may participate in the canalicular secretion of bicarbonate to bile.

Animals↗
PubMed2005

Dapsone impairs the bile salt-independent fraction of bile flow in rats: Possible involvement of its N-hydroxylated metabolite.

The effects of dapsone (DDS) treatment (30 mg/kg body wt, twice a day, for 4 days) on biliary secretory function, with special emphasis on bile salt independent bile flow (BSIF), were investigated in male and in female Wistar rats. Because DDS is metabolized to its N-hydroxylated parent compound only in male rats, any gender difference in DDS effect can be causally attributed to this metabolite. The two main driving forces for BSIF, the biliary secretion of HCO(3)(-) and glutathione species, were assessed. BSIF was decreased by about 20% in male but not in female rats after DDS treatment. Basal biliary HCO(3)(-) secretion was decreased also by 20% in males. This was associated with a diminished (-37%) expression of the HCO(3)(-) canalicular transporter, anion exchanger 2 (AE2), detected by western blotting. Biliary output of reduced glutathione (GSH) was not modified by DDS irrespective of gender, whereas excretion of oxidized glutathione (GSSG) was increased by 830% in males. This latter finding confirmed a gender-dependent oxidative stress associated with formation of the N-hydroxylated metabolite of DDS. The expression of multidrug resistance-associated protein 2 (Mrp2), a putative transporter of glutathione species, was decreased by 38% as detected by western blotting, clearly dissociating from preserved or increased biliary excretion of GSH and GSSG. In conclusion, our results show an impairment of BSIF by DDS mainly due to a decreased AE2-mediated biliary excretion of HCO(3)(-), formation of the N-hydroxylated metabolite of DDS being a likely mediator. The clinical relevance of these findings is discussed.

Animals↗
PubMed2003

Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type-dependent and correlates with the expression of Ank(195), a Golgi membrane skeletal protein.

Sodium-independent anion exchangers (AE1-4) show remarkable variability in their tissue-specific expression and subcellular localization. Currently, isoform-specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type-specific. We show that the full-length AE2 protein and its green fluorescent protein- or DsRed-tagged variants localize predominantly either to the Golgi apparatus in COS-7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post-translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin(195) (Ank(195)), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank(195) in the cells. The two Golgi-associated proteins also co-localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane-localized AE2 in Ank(195)-deficient cells was mostly detergent-soluble. Collectively, our results suggest that Ank(195) expression is a key determinant for the variable and cell type-dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.

Animals↗
PubMed1996

A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger.

Intracellular pH is maintained by a dynamic equilibrium balancing the opposing forces of proton loading and proton extrusion. By providing an efflux pathway for base, anion exchangers constitute a key component of the plasma membrane proton-loading machinery. The data in this paper identify a histidine-rich sequence within the cytoplasmic domain of the nonerythroid anion exchanger, AE2, that serves as an intracellular pH "sensor" that modulates anion exchange activity within the physiological range of cytoplasmic pH. These data reveal an interaction between the two major domains of the anion exchanger and suggest a novel substrate feedback mechanism by which intracellular protons directly control the activity of an acid-loading plasma membrane ion transporter.

Anion Transport Proteins↗
PubMed1998

Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells.

The hydrochloric acid secreting parietal cells of the human stomach mucosa have been shown to express anion exchanger 2 (AE2). AE2 is restricted to the basolateral membrane domain and is responsible for the basolateral uptake of Cl- and release of HCO3-. It is unknown which mechanism is responsible for the basolateral positioning of AE2 in parietal cells. We raised the question whether AE2 might be immobilized at the cell surface by linkage via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we communicate two observations that support this hypothesis, namely that in parietal cells ankyrin is localized with AE2 along the basolateral cell surface and, secondly, that purified erythrocyte ankyrin binds to the in vitro-translated cytoplasmic domain of AE2. We conclude from these observations that AE2 in parietal cells might be linked via ankyrin to the basolateral membrane cytoskeleton and that this type of linkage might play a role in immobilizing AE2 in a non-random fashion along the basolateral membrane domain.

Anion Transport Proteins↗
PubMed2000

Identification of the carbonic anhydrase II binding site in the Cl(-)/HCO(3)(-) anion exchanger AE1.

The human Cl(-)/HCO(3)(-) anion exchanger (AE1) possesses a binding site within its 33 residue carboxyl-terminal region (Ct) for carbonic anhydrase II (CAII). The amino acid sequence comprising this CAII binding site was determined by peptide competition and by testing the ability of truncation and point mutants of the Ct sequence to bind CAII with a sensitive microtiter plate binding assay. A synthetic peptide consisting of the entire 33 residues of the Ct (residues 879-911) could compete with a GST fusion protein of the Ct (GST-Ct) for binding to immobilized CAII, while a peptide consisting of the last 16 residues (896-911) could not. A series of truncation mutants of the GST-Ct showed that the terminal 21 residues of AE1 were not required for binding CAII. Removal of four additional residues (887-890) from the Ct resulted in loss of CAII binding. Acidic residues in this region (D887ADD) were critical for binding since mutating this sequence in the GST-Ct to DAAA, AAAA, or NANN caused loss of CAII binding. A GST-Ct construct mutated to D887ANE, the homologous sequence in AE2, could bind CAII. AE2 is a widely expressed anion exchanger and has a homologous Ct region with 60% sequence identity to AE1. A GST fusion protein of the 33 residue Ct of AE2 could bind to CAII similarly to the Ct of AE1. Tethering of CAII to an acidic motif within the Ct of anion exchangers may be a general mechanism for promoting bicarbonate transport across cell membranes.

Amino Acid Sequence↗
PubMed1998

Possible role of anion exchanger AE2 as the intestinal monocarboxylic acid/anion antiporter.

PURPOSE: The purpose of the present study was to investigate the transport of organic monocarboxylic acids mediated by the anion exchanger AE2, which has been already reported to be present at several tissue cell membranes, including intestinal brush border membrane in rabbit. METHODS: Membrane transport of organic monocarboxylic acids by AE2 was investigated by transient AE2-gene expression in HEK 293 cells and subsequent uptake studies by the cells. RESULTS: Functional transfection of AE2 was confirmed by the enhanced 36Cl- efflux from the cells. When preloaded with chloride anion. AE2-transfected cells demonstrated a significantly enhanced [14C]benzoic acid transport activity compared with mock-transfected cells. The AE2-mediated uptake was saturable with kinetic parameters of Km = 0.26 +/- 0.08 mM and Vmax = 6.14 +/- 0.52 nmol/mg protein/3 min and the uptake of [14C]benzoic acid was pH-dependent with a maximal uptake at pH 6.5. AE2-mediated [14C]benzoic acid uptake was inhibited by Cl-, HCO3-, and DIDS. AE2-transfected cells demonstrated significantly enhanced transport activity for nicotinic acid, propionic acid, butyric acid, and valproic acid as well as benzoic acid compared with mock-transfected cells. CONCLUSIONS: AE2 is functionally involved in the anion antiport for organic monocarboxylic acids as well as inorganic anions and is supposed to play a partial role in the intestinal transport of organic acids.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH.

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.

Acetates↗
PubMed1996

Three N-terminal variants of the AE2 Cl-/HCO3- exchanger are encoded by mRNAs transcribed from alternative promoters.

Multiple AE2 Cl-/HCO3- exchanger mRNAs have been identified in rat. To determine the genetic basis for these mRNAs and whether they encode different variants of the exchanger, we used both rapid amplification of cDNA ends and S1 nuclease protection protocols and examined the organization of the gene. mRNAs encoding three N-terminal variants of AE2 (AE2a, AE2b, and AE2c) were identified and shown to be transcribed from alternative promoters. The AE2a transcription unit consists of 23 exons, with exons 1 and 2 containing 5'-untranslated sequence and the first 17 codons. The first exon of AE2b is located in intron 2; it contains 5'-untranslated sequence and an alternative 3-amino acid N-terminal coding sequence and is spliced to exon 3. The first exon of AE2c is located in intron 5; it consists of 5'-untranslated sequence and is spliced to exon 6, which contains the translation initiation codon corresponding to Met-200 of AE2a. Northern analysis shows that AE2a is expressed in all tissues, AE2b exhibits a more restricted distribution with highest levels in stomach, and AE2c is expressed only in stomach. Thus, the use of alternative promoters leads to the production of three N-terminal variants of AE2 that exhibit tissue-specific patterns of expression.

Alternative Splicing↗
PubMed2004

Acute pH-dependent regulation of AE2-mediated anion exchange involves discrete local surfaces of the NH2-terminal cytoplasmic domain.

We have previously defined in the NH2-terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336-347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexa-Ala ((A)6) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312-578. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during changes in pHi or pHo in HEPES-buffered and in 5% CO2/HCO3- -buffered conditions. Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited at low pHo, with a pHo(50) value = 6.75 +/- 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336-347 attenuated pHi and/or pHo sensitivity of 36Cl- efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A)6357-362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His360. Homology modeling of the three-dimensional structure of the AE2 NH2-terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.

Amino Acid Sequence↗
PubMed2002

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger.

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na+-independent chloride-bicarbonate exchanger genes and multiple Na+-bicarbonate cotransporter and Na+-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na+-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.

Animals↗
PubMed1999

Transport activity of AE3 chloride/bicarbonate anion-exchange proteins and their regulation by intracellular pH.

Plasma membrane Cl(-)/HCO(3)(-) anion-exchange (AE) proteins contribute to regulation of intracellular pH (pH(i)). We characterized the transport activity and regulation by pH(i) of full-length AE3 and the cardiac isoform, AE3c, both of which are expressed in the heart. AE3c is an N-terminal variant of AE3. We also characterized AE1, AE2 and a deletion construct (AE3tr) coding for the common region of AE3 and AE3c. AE proteins were expressed by transient transfection of HEK-293 cells, and transport activity was monitored by following changes of intracellular pH or intracellular chloride concentration associated with anion exchange. Transport activities, measured as proton flux (mM H(+).min(-1)), were as follows: AE1, 24; AE2, 32; full-length AE3, 9; AE3c, 4 and AE3tr, 4. The wide range of transport activities is not explained by variation of cell surface processing since approx. 30% of each isoform was expressed on the cell surface. pH(i) was clamped at a range of values from 6.0-9.0 to examine regulation of AE proteins by pH(i). Whereas AE2 was steeply inhibited by acid pH(i), AE1, AE3 and AE3c were essentially insensitive to changes of pH(i). We conclude that AE3 and AE3c can contribute to pH(i) recovery after cellular-acid loading.

Animals↗
PubMed2006

Overexpression of anion exchanger 2 in human hepatocellular carcinoma.

To compare gene expression patterns between the poorly-differentiated (HA22T/VGH) and well-differentiated (HepG2) hepatocellular carcinoma cells, messenger RNA was isolated form both kinds of cells and subjected to differential displays reversing transcriptase-polymerase chain reaction (DDRT-PCR) technology. Gene fragments showing difference in the expression were recovered, reamplified, cloned and sequenced. Anion exchanger 2 (AE2), an isoform of band 3 protein, was identified and chosen for further characterization. AE2 was strongly expressed in HA22T/VGH cells, while it was little expressed in the well-differentiated hepatocellular carcinoma cells (PLC/PRF5 and HepG2) or little in the normal liver cell (Chang liver). In the 28 pairs of HCC and adjacent non-tumor tissues, levels in the cancer tissue (32.7 +/- 5.0) were significantly higher than that in the adjacent non-tumor tissue (9.1 +/- 2.4) (P < 0.01). Moreover, 19 cases (68%) showed over-expression of AE2 in HCC tissues, 3 cases were similar in both tissues, and 6 cases exhibited little or undetectable signals. Twenty cases (71%) of adjacent normal tissue showed little or undetectable signals. The results indicated that overexpression of AE2 may be involved in the development of human HCC.

Aged↗
PubMed1991

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line.

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

HNF1alpha upregulates the human AE2 anion exchanger gene (SLC4A2) from an alternate promoter.

The human AE2 gene (SLC4A2) is transcribed in a widespread fashion from the upstream promoter, the resultant full-length transcript AE2a being encountered in most tissues. Moreover, alternate promoter sequences within intron 2 may drive tissue-restricted expression of variants AE2b(1) and AE2b(2), mainly in liver and kidney. AE2b(2) proximal promoter sequences are highly active in transfected liver-derived HepG2 cells and contain an HNF1 motif. Mutation-disruption of this motif dramatically decreased alternate promoter activity in HepG2 cells but not in prostate-derived PC-3 cells. Electromobility shift and supershift assays indicated that HNF1alpha from HepG2 nuclear extracts binds the HNF1 sequence. Transactivation studies in PC-3 cells showed enhanced activity of the wild-type construct upon cotransfection with an HNF1alpha expression plasmid, while activity of the HNF1-mutated construct remained unaffected. Since liver AE2 is putatively involved in the biliary secretion of bicarbonate, HNF1alpha may have a role in increasing bicarbonate secretion in response to certain stimuli.

Amino Acid Motifs↗