Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W.

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl(-)/HCO(-) 3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.

Molecular cloning and functional characterization of zebrafish Slc4a3/Ae3 anion exchanger.

The zebrafish genome encodes two slc4a1 genes, one expressed in erythroid tissues and the other in the HR (H(+)-ATPase-rich) type of embryonic skin ionocytes, and two slc4a2 genes, one in proximal pronephric duct and the other in several extrarenal tissues of the embryo. We now report cDNA cloning and functional characterization of zebrafish slc4a3/ae3 gene products. The single ae3 gene on chromosome 9 generates at least two low-abundance ae3 transcripts differing only in their 5'-untranslated regions and encoding a single definitive Ae3 polypeptide of 1170 amino acids. The 7 kb upstream of the apparent initiator Met in ae3 exon 3 comprises multiple diverse, mobile repeat elements which disrupt and appear to truncate the Ae3 N-terminal amino acid sequence that would otherwise align with brain Ae3 of other species. Embryonic ae3 mRNA expression was detected by whole mount in situ hybridization only in fin buds at 24-72 hpf, but was detectable by RT-PCR across a range of embryonic and adult tissues. Epitope-tagged Ae3 polypeptide was expressed at or near the surface of Xenopus oocytes, and mediated low rates of DIDS-sensitive (36)Cl(-)/Cl(-) exchange in influx and efflux assays. As previously reported for Ae2 polypeptides, (36)Cl(-) transport by Ae3 was inhibited by both extracellular and intracellular acidic pH, and stimulated by alkaline pH. However, zebrafish Ae3 differed from Ae2 polypeptides in its insensitivity to NH4Cl and to hypertonicity. We conclude that multiple repeat elements have disrupted the 5'-end of the zebrafish ae3 gene, associated with N-terminal truncation of the protein and reduced anion transport activity.

Use of SLC26A4 mutation testing for unilateral enlargement of the vestibular aqueduct.

IMPORTANCE: Approximately one-half of all subjects with unilateral or bilateral hearing loss with enlargement of the vestibular aqueduct (EVA) will have SLC26A4 gene mutations. The number (0, 1, or 2) of mutant alleles of SLC26A4 detected in an individual subject with EVA is each associated with a distinct combination of diagnostic and prognostic information as well as probability of recurrence of EVA in siblings. OBJECTIVE: To evaluate the results of SLC26A4 mutation testing in subjects with unilateral EVA. (The study objective was formulated before data were collected.) DESIGN: Prospective cross-sectional study of cohort ascertained between 1998 and 2012. SETTING: National Institutes of Health Clinical Center, a federal biomedical research facility. PARTICIPANTS: Twenty-four subjects (10 males, 14 females) with unilateral EVA, defined as a midpoint diameter greater than 1.5 mm, who were referred or self-referred to participate in a study about the clinical and molecular analysis of EVA. Twenty-one (87.5%) of 24 subjects were white. Mean age was 10.3 years (age range, 5-39 years). INTERVENTION: SLC26A4 mutation analysis. MAIN OUTCOMES AND MEASURES: Audiometric results, the presence or absence of EVA, and the number of mutant alleles of SLC26A4. RESULTS: Approximately 8.3% of the subjects with unilateral EVA had 2 mutant SLC26A4 alleles, 16.7% had 1 mutant allele, and 75.0% had 0 mutant alleles. CONCLUSIONS AND RELEVANCE: Unilateral EVA can be associated with all possible SLC26A4 genotype results. The distinct combination of prognoses and recurrence probability associated with each genotype supports the clinical use of testing for SLC26A4 mutations in subjects with unilateral EVA.

Substitution of transmembrane domain Cys residues alters pH(o)-sensitive anion transport by AE2/SLC4A2 anion exchanger.

AE2/SLC4A2 is the most widely expressed of the Na(+)-independent SLC4 Cl(-)/HCO3 (-) exchangers and is essential for postnatal survival, but its structure remains unknown. We have generated and expressed a mouse AE2 construct devoid of transmembrane domain cysteine (Cys) residues, mAE2Cys-less, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2Cys-less expressed in Xenopus oocytes exhibited partial reduction of stilbene disulfonate-sensitive anion exchange activity. This activity was independent of the mAE2 N-terminal cytosolic domain and was accompanied by near-normal surface expression, without change in K 1/2 for extracellular Cl(-). mAE2Cys-less exhibited wildtype activation of anion exchange by hypertonicity and by NH4Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pHi) in the absence of NH4 (+). However, inhibition of anion exchange by extracellular pH (pHo) exhibited an alkaline shifted pHo(50) value of at least 0.6-0.7 pH units. Although SO4 (2-) transport by mAE2Cys-less resembled wildtype mAE2 in its stimulation by acidic pHo, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pHo. The contrasting enhancement of SO4 (2-) transport by alkaline pHo in the mAE1 anion translocation pathway mutant E699Q (Am J Physiol Cell Physiol 295: C302) was phenocopied by the corresponding mutant E1007Q in both AE2 and AE2Cys-less. However, the absence of transmembrane domain Cys residues exacerbated the reduced basal anion transport function exhibited by this and other missense substitutions at AE2 residue E1007. AE2Cys-less will be a valuable experimental tool for structure-function studies of the SLC4 gene family, but its utility for studies of AE2 regulation by extracellular pH must be evaluated in the context of its alkaline-shifted pHo sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.

Missense mutations in the ABCB6 transporter cause dominant familial pseudohyperkalemia.

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K(+)] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval identified-in 20 affected individuals among three multigenerational FP families-two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34(+) erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2-linked FP could contribute to the red cell K(+) leak characteristic of this condition.

Pendrin function and regulation in Xenopus oocytes.

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K((1/2)) (in mM) of 1.9 (Cl(-)), 1.8 (I(-)), and 0.9 (Br(-)). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization.

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO(3)(-) or more, mouse and rat ducts secrete ∼40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.

Regulated transport of sulfate and oxalate by SLC26A2/DTDST.

Nephrolithiasis in the Slc26a6(-/-) mouse is accompanied by 50-75% reduction in intestinal oxalate secretion with unchanged intestinal oxalate absorption. The molecular identities of enterocyte pathways for oxalate absorption and for Slc26a6-independent oxalate secretion remain undefined. The reported intestinal expression of SO(4)(2-) transporter SLC26A2 prompted us to characterize transport of oxalate and other anions by human SLC26A2 and mouse Slc26a2 expressed in Xenopus oocytes. We found that hSLC26A2-mediated [(14)C]oxalate uptake (K(1/2) of 0.65 +/- 0.08 mM) was cis-inhibited by external SO(4)(2-) (K(1/2) of 3.1 mM). hSLC26A2-mediated bidirectional oxalate/SO(4)(2-) exchange exhibited extracellular SO(4)(2-) K(1/2) of 1.58 +/- 0.44 mM for exchange with intracellular [(14)C]oxalate, and extracellular oxalate K(1/2) of 0.14 +/- 0.11 mM for exchange with intracellular (35)SO(4)(2-). Influx rates and K(1/2) values for mSlc26a2 were similar. hSLC26A2-mediated oxalate/Cl(-) exchange and bidirectional SO(4)(2-)/Cl(-) exchange were not detectably electrogenic. Both SLC26A2 orthologs exhibited nonsaturable extracellular Cl(-) dependence for efflux of intracellular [(14)C]oxalate, (35)SO(4)(2-), or (36)Cl(-). Rate constants for (36)Cl(-) efflux into extracellular Cl(-), SO(4)(2-), and oxalate were uniformly 10-fold lower than for oppositely directed exchange. Acidic extracellular pH (pH(o)) inhibited all modes of hSLC26A2-mediated anion exchange. In contrast, acidic intracellular pH (pH(i)) selectively activated exchange of extracellular Cl(-) for intracellular (35)SO(4)(2-) but not for intracellular (36)Cl(-) or [(14)C]oxalate. Protein kinase C inhibited hSLC26A2 by reducing its surface abundance. Diastrophic dysplasia mutants R279W and A386V of hSLC26A2 exhibited similar reductions in uptake of both (35)SO(4)(2-) and [(14)C]oxalate. A386V surface abundance was reduced, but R279W surface abundance was at wild-type levels.

Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

BACKGROUND: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. METHODOLOGY/PRINCIPAL FINDINGS: We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity. CONCLUSIONS/SIGNIFICANCE: The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter.

Oligomerization, function, and regulation of unmodified mouse Kcc1 K-Cl cotransporter were studied by chemical crosslinking. Treatment of Xenopus oocytes and 293T cells expressing K-Cl cotransporter Kcc1 with several types of chemical cross-linkers shifted Kcc1 polypeptide to higher molecular weight forms. More extensive studies were performed with the amine-reactive disuccinyl suberate (DSS) and with the sulfhydryl-reactive bis-maleimidohexane (BMH). Kcc1 cross-linking was time-dependent in intact oocytes, and was independent of protein concentration in detergent lysates from oocytes or 293T cells. Kcc1 cross-linking by the cleavable cross-linker DTME was reversible. The N-terminal and C-terminal cytoplasmic tails of Kcc1 were not essential for Kcc1 crosslinking. PFO-PAGE and gel filtration revealed oligomeric states of uncrosslinked KCC1 corresponding in mobility to that of cross-linked protein. DSS and BMH each inhibited KCC1-mediated (86)Rb(+) uptake stimulated by hypotonicity or by N-ethylmaleimide (NEM) without reduction in nominal surface abundance of KCC1. These data add to evidence supporting the oligomeric state of KCC polypeptides.

Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells.

Flow-induced cytosolic Ca2+ Ca(i)2+ signaling in renal tubular epithelial cells is mediated in part through P2 receptor (P2R) activation by locally released ATP. The ability of P2R to regulate salt and water reabsorption has suggested a possible contribution of ATP release and paracrine P2R activation to cystogenesis and/or enlargement in autosomal dominant polycystic kidney disease (ADPKD). We and others have demonstrated in human ADPKD cyst cells the absence of flow-induced Ca(i)2+ signaling exhibited by normal renal epithelial cells. We now extend these findings to primary and telomerase-immortalized normal and ADPKD epithelial cells of different genotype and of both proximal and distal origins. Flow-induced elevation of Ca(i)2+ concentration ([Ca2+](i)) was absent from ADPKD cyst cells, but in normal cells was mediated by flow-sensitive ATP release and paracrine P2R activation, modulated by ecto-nucleotidase activity, and abrogated by P2R inhibition or extracellular ATP hydrolysis. In contrast to the elevated ATP release from ADPKD cells in static isotonic conditions or in hypotonic conditions, flow-induced ATP release from cyst cells was lower than from normal cells. Extracellular ATP rapidly reduced thapsigargin-elevated [Ca2+](i) in both ADPKD cyst and normal cells, but cyst cells lacked the subsequent, slow, oxidized ATP-sensitive [Ca2+](i) recovery present in normal cells. Telomerase-immortalized cyst cells also exhibited altered CD39 and P2X7 mRNA levels. Thus the loss of flow-induced, P2R-mediated Ca(i)2+ signaling in human ADPKD cyst epithelial cells was accompanied by reduced flow-sensitive ATP release, altered purinergic regulation of store-operated Ca2+ entry, and altered expression of gene products controlling extracellular nucleotide signaling.

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis.

Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl(-), formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K(1/2) for sulfate of 4.0 muM. Na(+)-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWA-dependent sulfate transporter.

Mouse strain-specific coding polymorphism in the Slc4a2/Ae2 gene encodes Ae2c2 variants differing in isoform-specific dominant negative activity.

The Slc4a2/Ae2 gene encodes multiple polypeptides arising from alternate promoter usage. The Ae2c promoter gives rise to only one Ae2c transcript from the human Ae2 gene, but to two, alternatively spliced, Ae2c1 and Ae2c2 transcripts from the mouse and rat genes. Unlike in the rat, the mouse Ae2c2 transcript encodes a novel Ae2c2 polypeptide. Here we report that the Ae2c2 residue 9 can be either proline or serine in a mouse strain-specific manner. Both Ae2c2 polypeptides express low function in Xenopus oocytes secondary to reduced or absent surface expression. Ae2c2S, but not Ae2c2P, exerts a dominant negative effect when coexpressed with Ae2a polypeptide, has a less prominent effect when coexpressed with Ae2b1 or Ae2c1 polypeptides, but has no effect on the function of coexpressed Ae2b2 polypeptide. Coexpression of Ae2c2P does not reduce activity of any Ae2 polypeptide variant. Ae2c2S and Ae2c2P also express low functional activity in HEK-293 cells. Knowledge of strain-specific coding polymorphisms with potential functional consequences such as that of Ae2c2 should aid in interpretation of strain-specific phenotypes investigated in the mouse phenome project.

Zebrafish ae2.2 encodes a second slc4a2 anion exchanger.

The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene (Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. Am J Physiol Renal Physiol Renal Physiol 289: F835-F849, 2005), now called ae2.1. We now report the structural and functional characterization of Ae2.2, the product of the second zebrafish Ae2 gene, ae2.2. The ae2.2 gene of zebrafish linkage group 24 encodes a polypeptide of 1,232 aa in length, sharing 70% amino acid identity with zebrafish Ae2.1 and 67% identity with mouse AE2a. Zebrafish Ae2.2 expressed in Xenopus oocytes encodes a 135-kDa polypeptide that mediates bidirectional, DIDS-sensitive Cl(-)/Cl(-) exchange and Cl(-)/HCO3(-) exchange. Ae2.2-mediated Cl(-)/Cl(-) exchange is cation independent, voltage insensitive, and electroneutral. Acute regulation of anion exchange mediated by Ae2.2 includes activation by NH4+ and independent inhibition by acidic intracellular pH and by acidic extracellular pH. In situ hybridization reveals low-level expression of Ae2.2 mRNA in zebrafish embryo, most notably in posterior tectum, eye, pharynx, epidermal cells, and axial vascular structures, without notable expression in the Ae2.1-expressing pronephric duct. Knockdown of Ae2.2 mRNA, of Ae2.1 mRNA, or of both with nontoxic or minimally toxic levels of N-morpholino oligomers produced no grossly detectable morphological phenotype, and preserved normal structure of the head and the pronephric duct at 24 h postfertilization.

Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain.

The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.

Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization.

Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exon-intron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h postfertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates Na(+)-independent, electroneutral (36)Cl(-)/Cl(-) exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl(-)/HCO(3)(-) exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function.

Deficient HCO3- transport in an AE1 mutant with normal Cl- transport can be rescued by carbonic anhydrase II presented on an adjacent AE1 protomer.

Cl-/HCO3- exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of HCO3- to the intracellular HCO3- binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/HCO3- exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/HCO3- exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/HCO3- exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/HCO3- exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/HCO3- exchange activity in AE1 mutants selectively impaired in Cl-/HCO3- exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/HCO3- exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/HCO3- exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO3- transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.

Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes.

Mutations in the human SLC26A3 gene, also known as down-regulated in adenoma (hDRA), cause autosomal recessive congenital chloride-losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl--Cl- and Cl--HCO3- exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C-terminal amino acids from the putatively cytoplasmic C-terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti-sigma factor antagonist) domain abolished function. hDRA-mediated Cl- transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRA's N-terminal cytoplasmic tail or its 44 C-terminal amino acids, but they did require more proximate residues of the C-terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti-inflammatory drugs niflumate and tenidap. cAMP-insensitive Cl--HCO3- exchange mediated by hDRA gained modest cAMP sensitivity when co-expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild-type levels in proximal colon and nearly so in the distal ileum of CFTR(-/-) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.

Immunolocalization of potassium-chloride cotransporter polypeptides in rat exocrine glands.

Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at ca 135 kDa and ca 150 kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl(-) transport.