Regulation of anion exchanger Slc26a6 by protein kinase C

SLC26A6 (CFEX, PAT1) is an anion exchanger expressed in several tissues including renal proximal tubule, pancreatic duct, small intestine, liver, stomach, and heart. It has recently been reported that PKC activation inhibits A6-mediated Cl/HCO₃ exchange by disrupting binding of carbonic anhydrase to A6. However, A6 can operate in HCO₃-independent exchange modes of physiological importance, as A6-mediated Cl/oxalate exchange plays important roles in proximal tubule NaCl reabsorption and intestinal oxalate secretion. We therefore examined whether PKC activation affects HCO₃-independent exchange modes of Slc26a6 functionally expressed in Xenopus oocytes. We found that PKC activation inhibited Cl/formate exchange mediated by Slc26a6 but failed to inhibit the related anion exchanger pendrin (SLC26A4) under identical conditions. PKC activation inhibited Slc26a6-mediated Cl/formate exchange, Cl/oxalate exchange, and Cl/Cl exchange to a similar extent. The inhibitor sensitivity profile and the finding that PMA-induced inhibition was calcium independent suggested a potential role for PKC-. Indeed, the PKC--selective inhibitor rottlerin significantly blocked PMA-induced inhibition of Slc26a6 activity. Localization of Slc26a6 by immunofluorescence microscopy demonstrated that exposure to PKC activation led to redistribution of Slc26a6 from the oocyte plasma membrane to the intracellular compartment immediately below it. We also observed that PMA decreased the pool of Slc26a6 available to surface biotinylation but had no effect on total Slc26a6 expression. The physiological significance of these findings was supported by the observation that PKC activation inhibited mouse duodenal oxalate secretion, an effect blocked by rottlerin. We conclude that multiple modes of anion exchange mediated by Slc26a6 are negatively regulated by PKC- activation. oxalate; formate; chloride; duodenum     

Renal and intestinal transport defects in Slc26a6-null mice

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/high-Cl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P<0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P<0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by approximately 30% (P<0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum.     

Functional comparison of mouse slc26a6 anion exchanger with human SLC26A6 polypeptide variants: differences in anion selectivity, regulation, and electrogenicity

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.     

Decreased expression of Slc26a4 (Pendrin) and Slc26a7 in the kidneys of carbonic anhydrase II-deficient mice

BACKGROUND/AIMS: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice. METHODS: For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting. RESULTS: Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice. CONCLUSION: CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family - Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.     

Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6 null mice

Intestinal oxalate transport, mediated by anion exchange proteins, is important to oxalate homeostasis and consequently to calcium oxalate stone diseases. To assess the contribution of the putative anion transporter (PAT)1 (Slc26a6) to transepithelial oxalate transport, we compared the unidirectional and net fluxes of oxalate across isolated, short-circuited segments of the distal ileum of wild-type (WT) mice and Slc26a6 null mice [knockout (KO)]. Additionally, urinary oxalate excretion was measured in both groups. In WT mouse ileum, there was a small net secretion of oxalate (J(net)(Ox) = -5.0 +/-5.0 pmol.cm(-2).h(-1)), whereas in KO mice J(net)(Ox) was significantly absorptive (75 +/- 10 pmol.cm(-2)h.h(-1)), which was the result of a smaller serosal-to-mucosal oxalate flux (J(sm)(Ox)) and a larger mucosal-to-serosal oxalate flux (J(ms)(Ox)). Mucosal DIDS (200 microM) reduced J(sm)(Ox) in WT mice, leading to reversal of the direction of net oxalate transport from secretion to absorption (J(net)(Ox) = 15.0 +/- 5.0 pmol.cm(-2).h(-1)) , but DIDS had no significant effect on KO ileum. In WT mice in the absence of mucosal Cl(-), there were small increases in J(ms)(Ox) and decreases in J(sm)(Ox) that led to a small net oxalate absorption. In KO mice, J(net)(Ox) was 1.5-fold greater in the absence of mucosal Cl(-), due solely to an increase in J(ms)(Ox). Urinary oxalate excretion was about fourfold greater in KO mice compared with WT littermates. We conclude that PAT1 is DIDS sensitive and mediates a significant fraction of oxalate efflux across the apical membrane in exchange for Cl(-); as such, PAT1 represents a major apical membrane pathway mediating J(sm)(Ox).     

Deletion of multispecific organic anion transporter Oat1/Slc22a6 protects against mercury-induced kidney injury

The primary site of mercury-induced injury is the kidney due to uptake of the reactive Hg(2+)-conjugated organic anions in the proximal tubule. Here, we investigated the in vivo role of Oat1 (organic anion transporter 1; originally NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478)) in handling of known nephrotoxic doses of HgCl(2). Oat1 (Slc22a6) is a multispecific organic anion drug transporter that is expressed on the basolateral aspects of renal proximal tubule cells and that mediates the initial steps of elimination of a broad range of endogenous metabolites and commonly prescribed pharmaceuticals. Mercury-induced nephrotoxicity was observed in a wild-type model. We then used the Oat1 knock-out to determine in vivo whether the renal injury effects of mercury are mediated by Oat1. Most of the renal injury (both histologically and biochemically as measured by blood urea nitrogen and creatinine) was abolished following HgCl(2) treatment of Oat1 knock-outs. Thus, acute kidney injury by HgCl(2) was found to be mediated mainly by Oat1. Our findings raise the possibility that pharmacological modulation of the expression and/or function of Oat1 might be an effective therapeutic strategy for reducing renal injury by mercury. This is one of the most striking phenotypes so far identified in the Oat1 knock-out. (Eraly, S. A., Vallon, V., Vaughn, D. A., Gangoiti, J. A., Richter, K., Nagle, M., Monte, J. C., Rieg, T., Truong, D. M., Long, J. M., Barshop, B. A., Kaler, G., and Nigam, S. K. (2006) J. Biol. Chem. 281, 5072-5083).     

Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3--buffered Cl--free media. These findings were used to develop a turnover cycle for Cl- and HCO3- transport by slc26a3. Cl- and HCO3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl--mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl- absorption and HCO3- secretion.     

Characterization and regulation of monocarboxylate cotransporters Slc16a7 and Slc16a3 in preimplantation mouse embryos

Concurrent with compaction, preimplantation mouse embryos switch from the high pyruvate consumption that prevailed during cleavage stages to glucose consumption against a constant background of pyruvate uptake. However, zygotes exposed to and subsequently deprived of glucose can form blastocysts by increasing pyruvate uptake. This metabolic switch requires cleavage-stage exposure to glucose and is one aspect of metabolic differentiation that normally occurs in vivo. Monocarboxylates, such as pyruvate and lactate, are transported across membranes via the SLC16 family of H(+)-monocarboxylate cotransporter (MCT) proteins. Thus, the increase in pyruvate uptake in embryos developing without glucose must involve changes in activity and localization of MCT. In mouse embryos, continued expression of Slc16a1 (MCT1) requires glucose supply. Messenger RNA for Slc17a7 (MCT2) and Slc16a3 (MCT4) has been detected in mouse preimplantation embryos; however, protein function, localization, and regulation of expression at the basis of these net pyruvate uptake changes remain unclear. The expression and localization of SLC16A7 and SLC16A3 have therefore been examined to clarify their respective roles in embryos derived from the reproductive tract and cultured under varied conditions. SLC16A3 appears localized to the plasma membrane until the morula stage and also maintains a nuclear distribution throughout preimplantation development. However, continued Slc16a3 mRNA expression is dependent on prior exposure to glucose. SLC16A7 localizes to apical cortical regions with punctate, vesicular expression throughout blastomeres, partially colocalizing in peroxisomes with peroxisomal catalase (CAT). In contrast to SLC16A3 and SLC16A1, SLC16A7 and CAT demonstrate upregulation in the absence of glucose. These striking differences between the two isoforms in expression localization and regulation suggest unique roles for each in monocarboxylate transport and pH regulation during preimplantation development, and implicate peroxisomal SLC16A7 as an important redox regulator in the absence of glucose.     

Oligonucleotide analysis by anion exchange HPLC.

The ability to resolve and purify synthetic oligonucleotides by high performance anion exchange chromatography was evaluated using two wide pore polymeric HPLC matrices. The materials used are rigid macroporous copolymers which have a fully quaternised polyethyleneimine coating to provide a strong anion exchange, quaternary amine, functionality. Oligomers of poly(rA), poly(rC) and RNA produced by alkaline hydrolysis of the polymers were chromatographed to evaluate the selectivity of the system prior to the analysis of synthetic oligonucleotides produced using a commercial oligonucleotide synthesizer.     

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.     

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes

A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.     

Slc4-like anion transporters of the larval mosquito alimentary canal

Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH>10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na(+)-independent chloride/bicarbonate anion exchanger (AE) as well as a Na(+)-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.     

Uronic acid analysis by automated anion exchange chromatography

A rapid and sensitive assay for individual uronic acids has been developed based on their separation on a 0.5 × 22-cm column of Aminex A-25 in 0.12 m Tris-acetate buffer, pH 7.4. Quantification of these sugars is accomplished by coupling the column to the analytical portion of a Technicon sugar analyzer. Each determination is complete in 3 hr, and as little as 25 nmol of uronic acid can be measured with accuracy.     

Heterogeneity of lipoteichoic acid detected by anion exchange chromatography

A complex polydispersity became apparent when the poly(glycerophosphate) lipoteichoic acid of Enterococcus faecalis was chromatographed on DEAE-sephadex. The chain length varied between 13 and 33 glycerophosphate residues per lipid anchor. In parallel, the extent of chain glycosylation increased from 0.2 to 0.4 diglucosyl residues per glycerophosphate unit. Substitution with D-alanine ester showed a reverse distribution dropping with increasing chain length from 0.53 to 0.23 mol D-alanine per mol phosphorus. Variations in the fatty acid composition were also observed. The results extent and modify the current picture of lipoteichoic acid biosynthesis. They further suggest that during infection the mammalian organism may be confronted particularly with long-chain less hydrophobic molecular species.     

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.     

Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger

Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.     

Specificity in transmembrane helix-helix interactions mediated by aromatic residues

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through pi-pi interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458-15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.