Molecular aspects of renal tubular handling and regulation of inorganic sulfate
2001; Elsevier BV; Volume: 59; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1755.2001.059003835.x
ISSN1523-1755
Autores Tópico(s)Renal function and acid-base balance
ResumoMolecular aspects of renal tubular handling and regulation of inorganic sulfate.The renal proximal tubular reabsorption of sulfate plays an important role in the maintenance of sulfate homeostasis. Two different renal sulfate transport systems have been identified and characterized at the molecular level in the past few years: NaSi-1 and Sat-1. NaSi-1 belongs to a Na+-coupled transporter family comprising the Na+-dicarboxylate transporters and the recently characterized SUT1 sulfate transporter. NaSi-1 is a Na+-sulfate cotransporter located exclusively in the brush border membrane of renal proximal tubular and ileal cells. Recently, NaSi-1 was shown to be regulated at the protein and mRNA level by a number of factors, such as vitamin D, dietary sulfate, glucocorticoids and thyroid hormones, which are known to modulate sulfate reabsorption in vivo. The second member of renal sulfate transporters, denoted Sat-1, belongs to a family of Na+-independent sulfate transporter family comprising the DTDST, DRA and PDS genes. Sat-1 is a sulfate/bicarbonate-oxalate exchanger located at the basolateral membrane of proximal tubular epithelial cells and canalicular surface of hepatic cells. Contrary to NaSi-1, no physiological factor has been found to date to regulate Sat-1 gene expression. Both NaSi-1 and Sat-1 transporter activities are implicated in pathophysiological states such as heavy metal intoxication and chronic renal failure. This review focuses on recent developments in the molecular characterization of NaSi-1 and Sat-1 and the mechanisms involved in their regulation. Molecular aspects of renal tubular handling and regulation of inorganic sulfate. The renal proximal tubular reabsorption of sulfate plays an important role in the maintenance of sulfate homeostasis. Two different renal sulfate transport systems have been identified and characterized at the molecular level in the past few years: NaSi-1 and Sat-1. NaSi-1 belongs to a Na+-coupled transporter family comprising the Na+-dicarboxylate transporters and the recently characterized SUT1 sulfate transporter. NaSi-1 is a Na+-sulfate cotransporter located exclusively in the brush border membrane of renal proximal tubular and ileal cells. Recently, NaSi-1 was shown to be regulated at the protein and mRNA level by a number of factors, such as vitamin D, dietary sulfate, glucocorticoids and thyroid hormones, which are known to modulate sulfate reabsorption in vivo. The second member of renal sulfate transporters, denoted Sat-1, belongs to a family of Na+-independent sulfate transporter family comprising the DTDST, DRA and PDS genes. Sat-1 is a sulfate/bicarbonate-oxalate exchanger located at the basolateral membrane of proximal tubular epithelial cells and canalicular surface of hepatic cells. Contrary to NaSi-1, no physiological factor has been found to date to regulate Sat-1 gene expression. Both NaSi-1 and Sat-1 transporter activities are implicated in pathophysiological states such as heavy metal intoxication and chronic renal failure. This review focuses on recent developments in the molecular characterization of NaSi-1 and Sat-1 and the mechanisms involved in their regulation. Inorganic sulfate, one of the most abundant anions in mammalian plasma, is a highly dissociated, hydrophilic, divalent anion required for normal cellular function. Sulfate is involved in many physiological processes and in particular is utilized in the sulfation of a variety of exogenous and endogenous compounds1Mulder G.J. Sulfate availability in vivo,.Sulfation of Drugs and Related Compounds. edited by Mulder GJ. CRC, Boca Raton1981: 32-52Google Scholar. Sulfate conjugation of xenobiotics such as steroids, anti-inflammatory agents, and adrenergic blockers and stimulants, results in their biotransformation. In most cases, the sulfation of these compounds leads to an increase in their urinary excretion2Falany C.N. Enzymology of human cytosolic sulfotransferases.FASEB J. 1997; 11: 206-216Crossref PubMed Scopus (520) Google Scholar. Sulfation also activates a number of endogenous compounds such as heparin, gastrin, cholecystokinin, and heparan sulfate. Finally, sulfate is also necessary for the biosynthesis of structural components of membranes and tissues, such as sulfated glycosaminoglycans present in proteoglycans. Proteoglycans represent the largest group of sulfoconjugates, and proteoglycan sulfation is essential to maintain their properties required for the maintenance of normal structure and function of tissues. This has been strikingly demonstrated by the identification of mutations in a sulfate transport protein gene (DTDST, for DiasTrophic Dysplasia Sulfate Transporter) in three different types of osteochondrodysplasias, diastrophic dysplasia, type IB achodrongenesis, and type II atelosteogenesis3Hastbacka J. Superti-Furga A. Wilcox W.R. Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): Evidence for a phenotypic series involving three chondrodysplasias.Am J Human Genet. 1996; 58: 255-262PubMed Google Scholar, 4Hästbacka J. de la Chapelle A. Mahtani M. The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (622) Google Scholar, 5Superti-Furga A. A defect in the metabolic activation of sulfate in a patient with achondrogenesis type IB.Am J Human Genet. 1994; 55: 1137-1145PubMed Google Scholar. Each of these chondrodysplasias is associated with undersulfation of cartilage proteoglycans. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein, that is, on the intracellular sulfate concentration. Sulfation of all naturally occurring sulfated compounds, including proteoglycans, depends on the availability of intracellular inorganic sulfate concentration. The inorganic form of sulfate account for 90 to 95%, whereas sulfoconjugates represent only 5 to 10% of total body sulfate. Inorganic sulfate is the precursor of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the activated form of sulfate and substrate of the sulfotransferases6White A. Handler P. Smith E.L. Amino acid metabolism. II,.Principles of Biochemistry. edited by White A Handler P Smith EL. McGraw-Hill, New York1968: 563-566Google Scholar. Intracellular inorganic sulfate concentration depends on the activity of specific sulfate transporters (such as DTDST) and on extracellular inorganic sulfate concentration. In this regard, we have recently demonstrated that vitamin D status modulates plasma sulfate concentrations, and as a result, may influence the amount of inorganic sulfate available for intracellular sulfation of proteoglycans. Furthermore, altered proteoglycan synthesis by aortic endothelial cells7Humphries D.E. Silbert C.K. Silbert J.E. Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium.J Biol Chem. 1986; 261: 9122-9127Abstract Full Text PDF PubMed Google Scholar and epiphyseal cartilage8Ito K. Kimata K. Sobue M. Suzuki S. Altered proteoglycan synthesis by epiphyseal cartilages in culture at low SO4(2-) concentration.J Biol Chem. 1982; 257: 917-923Abstract Full Text PDF PubMed Google Scholar maintained in vitro in medium containing low sulfate concentrations has been observed. Thus, a limitation in the availability of inorganic sulfate produced by acquired or inherited defects may also result in abnormal sulfation of sulfoconjugates. In spite of its clear importance, sulfate levels are almost never measured in clinical practice, little is known about the factors that regulate sulfate homeostasis in mammals, and the possible implication of alteration in sulfate metabolism in the pathogenesis of human diseases is largely unexplored. Sulfate requirements in human and other mammalian species appear to change as a function of growth and age. Plasma sulfate concentration has been reported to decrease as a function of age (from 0.47 mmol/L at birth to 0.33 mmol/L in adulthood)9Tallgren L. Inorganic sulphate in relation to the serum thyroxine level and in renal failure.Acta Med Scand. 1980; Suppl 640: 1-100Google Scholar, 10Cole D. Scriver C. Age dependent serum sulfate levels in children and adolescents.Clin Chim Acta. 1980; 107: 135-139Crossref PubMed Scopus (31) Google Scholar, 11Bakhtian S. Kimura R. Galinsky R. Age-related changes in homeostasis of inorganic sulfate in male F-344 rats.Mech Ageing Dev. 1993; 66: 257-267Crossref Scopus (11) Google Scholar, 12Pena D. Neiberger R. Renal brush border sodium-sulfate cotransport in guinea-pig: effect of age and diet.Pediatr Nephrol. 1997; 11: 724-727Crossref Scopus (8) Google Scholar. A slight decrease has been described during puberty, while a slight increase occurs after 45 and 75 years in men and women, respectively. Values do not vary significantly between sex. In women, variations in sulfatemia during the menstrual cycle (increase during the first two weeks of the cycle with a peak at 14 and 15 days) and during the menopause (decrease) have been reported13Benincosa L.J. Sagawa K. Massey L.K. Morris M.E. Effects of acute caffeine ingestion and menopause on sulfate homeostasis in women.Life Sci. 1995; 57: 1497-1505Crossref Scopus (10) Google Scholar. Sulfatemia is increased in women in the third trimester of pregnancy. Body sulfate homeostasis results from the balance between diet, intestinal absorption and renal excretion. Sulfate supply from the diet can vary from 1.5 to 16 mmol of sulfate per 24 hours14Florin T. Neale G. Gibson G. Metabolism of dietary sulphate: Absorption and excretion in humans.Gut. 1991; 32: 766-773Crossref PubMed Scopus (171) Google Scholar, from which 90 to 95% are absorbed along the intestine, mainly in the distal ileum9Tallgren L. Inorganic sulphate in relation to the serum thyroxine level and in renal failure.Acta Med Scand. 1980; Suppl 640: 1-100Google Scholar. Sulfate transport on the apical side of ileal enterocytes is mediated by a high affinity low capacity Na+-dependent sulfate transporter14Florin T. Neale G. Gibson G. Metabolism of dietary sulphate: Absorption and excretion in humans.Gut. 1991; 32: 766-773Crossref PubMed Scopus (171) Google Scholar, 15Lücke H. Stange G. Murer H. Sulfate-sodium cotransport by brush-border membrane vesicles isolated from rat ileum.Gastroenterology. 1981; 80: 22-30Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 16Ahearn G.A. Murer H. Functional roles of Na+ and H+ in SO42- transport by rabbit ileal brush border membrane vesicles.J Membr Biol. 1984; 78: 177-186Crossref PubMed Scopus (39) Google Scholar. Transport of sulfate from the cell to the extracellular compartment through the basolateral membrane is thought to be mediated by a sulfate/chloride exchanger17Langridge-Smith J. Field M. Sulfate transport in rabbit ileum: characterization of the serosal border anion exchange process.J Membr Biol. 1981; 63: 207-214Crossref PubMed Scopus (34) Google Scholar, 18Schron C.M. Knickelbein R.G. Aronson P.S. Dobbins J.W. Evidence for carrier-mediated Cl-SO4 exchange in rabbit ileal basolateral membrane vesicles.Am J Physiol. 1987; 253: G404-G410PubMed Google Scholar, 19Hagenbush B. Stange G. Murer H. Transport of sulphate in rat jejunal and proximal tubular basolateral membrane vesicles.Pflügers Arch. 1985; 405: 202-208Crossref Scopus (49) Google Scholar. Despite large variations of intestinal sulfate intake, plasma sulfate concentration is fairly constant over a 24 hour period20Hoffman D. Wallace S. Verbeeck R. Circadian rhythm of serum sulfate levels in man and acetaminophen pharmacocinetics.Eur J Clin Pharmacol. 1990; 39: 143-148Crossref Scopus (23) Google Scholar. In humans, the maintenance of sulfate homeostasis is largely determined by the kidney. Sulfate is freely filtered at the glomerulus and reabsorbed in the proximal tubule. Under physiological conditions, tubular sulfate reabsorption works near the maximal rate, with only 5 to 20% of the filtered load being excreted in the urine21Mudge G. Berndt W. Valtin H. Tubular transport of urea, phosphate, uric acid, sulfate and thiosulfate,.Handbook of Physiology. edited by Orloff B. Geiger, Washington DC1973: 587-652Google Scholar. If plasma sulfate concentration increases, the filtered load of sulfate quickly exceeds the maximal tubular reabsorption and non-reabsorbed sulfate is highly excreted in the urine. Transepithelial transport of sulfate from the renal lumen to the blood compartment involves entry through the brush border membrane (BBM) by a Na+-dependent transport system22Lücke H. Stange G. Murer H. Sulphate/ion-sodium/ion co-transport by brush border membranes vesicles isolated from rat kidney cortex.Biochem J. 1979; 182: 223-229Crossref PubMed Scopus (89) Google Scholar, 23Schneider E.G. Durham J.C. Sacktor B. Sodium-dependent transport of inorganic sulfate by rabbit renal brush-border membrane vesicles.J Biol Chem. 1984; 259: 14591-14599Abstract Full Text PDF PubMed Google Scholar, 24Turner R. Sodium-dependent sulfate transport in renal outer cortical brush border membrane vesicles.Am J Physiol. 1984; 247: F793-F798PubMed Google Scholar, 25David C. Ullrich K. Substrate specificity of the luminal Na(+)-dependent sulphate transport system in the proximal renal tubule as compared to the contraluminal sulphate exchange system.Pflügers Arch. 1992; 421: 455-465Crossref Scopus (22) Google Scholar, translocation across the cell, and finally efflux across the basolateral membrane by an anion exchange mechanism19Hagenbush B. Stange G. Murer H. Transport of sulphate in rat jejunal and proximal tubular basolateral membrane vesicles.Pflügers Arch. 1985; 405: 202-208Crossref Scopus (49) Google Scholar,26Pritchard J. Renfro J. Renal sulfate transport at the basolateral membrane is mediated by anion exchange.Proc Natl Acad Sci USA. 1983; 80: 2603-2607Crossref PubMed Scopus (88) Google Scholar. In recent years, two main families of sulfate transporters have been identified in vertebrates. The first family comprises Na+-coupled SO42- transporters, the prototype of which is NaSi-1, expressed in the renal proximal tubule. SUT-1, another member of this family, has been recently identified and shown to be expressed in veinules in the placenta. The second family comprises anion exchangers, the prototype of which is a sulfate/oxalate-bicarbonate anion exchanger (Sat-1). Sat-1 was first isolated by expression cloning from a rat liver cDNA library, and subsequently shown to be expressed in the renal proximal tubule. The other members of this family are the ubiquitously expressed DTDST, discussed above, and the DRA (down regulated in adenoma), a chloride-sulfate exchanger expressed in the colon. The NaSi-1 and Sat-1 transporters have been localized to the apical and basolateral membrane of proximal tubular epithelial cells, respectively Figure 127Karniski L. Lotscher M. Fucentese M. Immunolocalisation of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney.Am J Physiol. 1998; 275: F79-F87PubMed Google Scholar, 28Custer M. Murer H. Biber J. Nephron localization of Na/SO4(2-)-cotransport-related mRNA and protein.Pflügers Arch. 1994; 429: 165-168Crossref PubMed Scopus (19) Google Scholar, 29Lotscher M. Custer M. Quabius E.S. Immunolocalization of Na/SO4- cotransport (NaSi-1) in rat kidney.Pflügers Arch. 1996; 432: 373-378Crossref PubMed Scopus (48) Google Scholar, and have been hypothetized to play a major role in the regulation of body sulfate homeostasis by mediating sulfate entry and exit from the proximal tubular cells. In the present review, we will discuss the molecular identification and properties of NaSi-1 and Sat-1, their physiological regulation, and their implication in different pathophysiological states. Isolation of the first mammalian sulfate transporter was achieved in 1993 by the use of the Xenopus oocytes expression cloning system30Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar. This transporter, isolated from a rat kidney cortex cDNA library, was shown to encode a Na+-coupled sulfate transporter and was named NaSi-1 for Na-sulfate cotransporter-130Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar. Northern blot analysis shows signals of 2.3 and 2.9 kb in rat kidney and ileum. The NaSi-1 transporter expressed in ileum has been isolated by homology cloning and appears to be identical to the renal NaSi-131Norbis F. Perego C. Markovich D. cDNA cloning of a rat small-intestinal Na+/SO4(2-) cotransporter.Pflügers Arch. 1994; 428: 217-223Crossref Scopus (18) Google Scholar. The NaSi-1 cDNA encodes for a protein of 595 amino acids (66 kD). In vitro translation of NaSi-1 cRNA results in a protein of the expected size and suggests glycosylation. Recently, we identified homologous proteins in mouse32Beck L. Markovich D. The mouse Na+-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D.J Biol Chem. 2000; 275: 11800-11890Google Scholar and humans (unpublished data). The mouse (mNaSi-1) and human (hNaSi-1) NaSi-1 cDNAs encode proteins sharing 94% and 83% identity with the rat NaSi-1 (rNaSi-1), respectively. Secondary structure prediction of the NaSi-1 protein based on hydropathy analysis and the inside positive rule predicts the presence of 13 transmembrane segments Figure 2. The N-terminal leader does not contain any discernible signal sequence and is predicted to be inside the cell, whereas the COOH terminus is placed extracellularly. This model contrasts with the secondary structure prediction of rNaSi-1 protein, which was initially predicted to contain eight transmembrane segments30Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar. The difference is not due to a different protein structure based on amino acid sequence, but due to the prediction method used. However, it should be noted that direct experimental evidence for the existence of such membrane topologies is missing. The NaSi-1 proteins contain three (rat and mouse) or four (human) consensus sites for N-linked glycosylation. However, due to the predicted secondary structure of NaSi-1, only the Asn residue located at the C-terminus, is suggested to be extracellular Figure 2. If exact, this observation would be consistent with previous findings showing that core glycosylation led to a modest 3 to 4 kD upward shift on SDS-PAGE, suggesting that N-glycosylation occurs on only one of the three putative N-glycosylation sites of rNaSi-130Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar. NaSi-1 also contains one putative protein kinase A and four (human) or five (mouse/rat) putative protein kinase C phosphorylation sites, for which the functional significance still needs to be determined. Homology searches of protein sequence databases with NaSi-1 protein reveals significant homology to approximately 20 other proteins. These sequences are found across a large taxonomic span—including animals, plants, yeast and bacteria—although the closest relatives are the recently reported human Na+-sulfate cotransporter SUT-1 (48% identity)33Girard J.P. Baekkevold E.S. Feliu J. Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules.Proc Natl Acad Sci USA. 1999; 96: 12772-12777Crossref PubMed Scopus (53) Google Scholar and the Na+-dicarboxylate cotransporters (NaDC and SDCT) sharing ∼32 to 43% protein sequence identity with NaSi-1 Table 1. Of particular interest is a consensus pattern previously established for Na+-coupled symporters (PROSITE PS01271) known as the "Na+-sulfate signature," present in the carboxy terminal region of the proteins and showing a very high degree of homology among the related proteins Table 1. The exact function of this region is yet to be determined. However, it is interesting to note that protein chimeras containing the first four transmembrane domains of NaSi-1 and the last seven transmembrane domains of NaDC-1 retained the substrate selectivity for NaDC-1, but not for NaSi-134Pajor A.M. Sun N. Bai L. The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein.Biochim Biophys Acta. 1998; 1370: 98-106Crossref PubMed Scopus (30) Google Scholar. Along the same line, the C-terminal half of the Na+-glucose cotransporters SGLT1 and SGLT2 were shown to contain residues that determine substrate affinity35Panayotova-Heiermann M. Loo D.D. Kong C.T. Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.J Biol Chem. 1996; 271: 10029-10034Crossref PubMed Scopus (93) Google Scholar. It is therefore possible that the highly conserved "Na+-sulfate signature" present in NaSi-1 plays an important role for substrate specificity.Table 1The mammalian Na+-sulfate/Na+-dicarboxylate cotransporter gene familyGeneSpeciesFunctionProtein sizeTransmembrane segmentsaDetermination of the secondary structure was performed using the TopPred2 program. Numbers refer to the number of transmembrane segments determined with certainty.Overall identitybIdentity to the full-length rat NaSi-1 protein, as determined with the aligment program ClustalW%Symporter signature identitycIdentity to the Na+-sulfate motif (Prosite PS01271) found in rat NaSi-1 protein, as determined with the aligment program ClustalW%GenBank accession numberrNaSi-1RatRenal Na+-sulfate transporter, identical to ileal rNaSi-1 (U08031)59513100100L19102mNaSi-1MouseRenal and ileal Na+-sulfate transporter5941393.6100AF199365hNaSi-1HumanRenal Na+-sulfate transporter5951382.9100AF260824hSUT-1HumanHigh endothelial venule Na+-sulfate transporter627124876.5AF169301xNaDC-2Xenopus laevisIntestinal sodium/dicarboxylate cotransporter6221343.270.6U87318hNaDC-1HumanRenal sodium/dicarboxylate cotransporter5921342.282.4U26209rSDCT1RatRenal sodium/dicarboxylate cotransporter5871339.676.5AF058714.1rbNaDC-1RabbitRenal sodium/dicarboxylate cotransporter5931339.676.5U12186rNaDC-1RatRenal sodium/dicarboxylate cotransporter5871338.170.6AB001321rSDCT2RatPlacental sodium/dicarboxylate cotransporter, identical to rNaDC36001336.358.8AF080451.1rNaDC3RatRenal sodium/dicarboxylate cotransporter, identical to rSDCT26001336.358.8AF081825.1fNaDC3Winter flounderRenal sodium/dicarboxylate cotransporter601133558.8AF102261.1rNaDC-2RatIntestinal sodium/dicarboxylate cotransporter5871231.976.5U51153a Determination of the secondary structure was performed using the TopPred2 program. Numbers refer to the number of transmembrane segments determined with certainty.b Identity to the full-length rat NaSi-1 protein, as determined with the aligment program ClustalWc Identity to the Na+-sulfate motif (Prosite PS01271) found in rat NaSi-1 protein, as determined with the aligment program ClustalW Open table in a new tab Phylogenetic analysis shows that human, mouse and rat NaSi-1s are closely related, and that together with SUT-1 constitute a distinct protein family belonging to a superfamily of Na+-coupled membrane proteins that include the Na+-sulfate and Na+-dicarboxylate cotransporters. The NaSi-1 transporter has been identified on the basis of its Na+-dependent sulfate transport function30Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar. The transport properties of the different NaSi-1 transporters have been studied in oocytes, in different transfected cell lines expressing the transporter and by electrophysiological techniques with oocytes32Beck L. Markovich D. The mouse Na+-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D.J Biol Chem. 2000; 275: 11800-11890Google Scholar, 36Busch A.E. Waldegger S. Herzer T. Electrogenic cotransport of Na+ and sulfate in Xenopus oocytes expressing the cloned Na+-SO4(2-) transport protein NaSi-1.J Biol Chem. 1994; 269: 12407-12409Abstract Full Text PDF PubMed Google Scholar, 37Quabius E.S. Murer H. Biber J. Expression of proximal tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLC-PK1 cells by transfection.Am J Physiol. 1996; 270: F220-F228PubMed Google Scholar, 38Markovich D. Bissig M. Sorribas V. Expression of rat renal sulfate transport systems in Xenopus laevis oocytes. Functional characterization and molecular identification.J Biol Chem. 1994; 269: 3022-3026Abstract Full Text PDF PubMed Google Scholar. The NaSi-1 transporter leads to an augmented Na+-dependent tracer sulfate uptake when expressed in either oocytes or Madin-Darby canine kidney (MDCK) cells. The characteristics of the NaSi-1-mediated Na+-sulfate cotransport is summarized in Table 2. These data, and in particular the work of Busch et al36Busch A.E. Waldegger S. Herzer T. Electrogenic cotransport of Na+ and sulfate in Xenopus oocytes expressing the cloned Na+-SO4(2-) transport protein NaSi-1.J Biol Chem. 1994; 269: 12407-12409Abstract Full Text PDF PubMed Google Scholar, demonstrate that the stoichiometry of the transport process is 3:1 for Na+-sulfate cotransport Figure 1, leading to electrogenicity. NaSi-1-induced sulfate transport is inhibited by thiosulfate, selenate, molybdate and tungstate, whereas phosphate has no effect on this activity30Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar, 32Beck L. Markovich D. The mouse Na+-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D.J Biol Chem. 2000; 275: 11800-11890Google Scholar, 38Markovich D. Bissig M. Sorribas V. Expression of rat renal sulfate transport systems in Xenopus laevis oocytes. Functional characterization and molecular identification.J Biol Chem. 1994; 269: 3022-3026Abstract Full Text PDF PubMed Google Scholar. Moreover, we recently observed that succinate and citrate are able to inhibit hNaSi-1 induced sulfate transport, whereas succinate and oxalate are not transported by hNaSi-1 (unpublished data). This suggests that dicarboxylates can act as competitive inhibitors for sulfate binding on NaSi-1, but are not direct substrates for transport, in agreement with a recent study showing the inability of rNaSi-1 to transport succinate34Pajor A.M. Sun N. Bai L. The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein.Biochim Biophys Acta. 1998; 1370: 98-106Crossref PubMed Scopus (30) Google Scholar. Finally, the absence of transport activity of L-arginine, L-leucine or D-glucose by NaSi-1, the inability for the anion-exchange inhibitor DIDS to block NaSi-1 activity, and the lack of pH dependence30Markovich D. Forgo J. Stange G. Expression cloning of rat renal Na+/SO42- cotransport.Proc Natl Acad Sci USA. 1993; 90: 8073-8077Crossref PubMed Scopus (145) Google Scholar, 31Norbis F. Perego C. Markovich D. cDNA cloning of a rat small-intestinal Na+/SO4(2-) cotransporter.Pflügers Arch. 1994; 428: 217-223Crossref Scopus (18) Google Scholar, 38Markovich D. Bissig M. Sorribas V. Expression of rat renal sulfate transport systems in Xenopus laevis oocytes. Functional characterization and molecular identification.J Biol Chem. 1994; 269: 3022-3026Abstract Full Text PDF PubMed Google Scholar, suggest that NaSi-1 encodes a Na+-coupled sulfate transporter, whose function correlates with the BBM Na+-dependent symporter of proximal tubular cells39Murer H. Manganel M. Roch-Ramel F. Tubular transport of monocarboxylates, Krebs cycle intermediates and inorganic sulphate,.Handbook of Physiology. edited by Winhager E. Oxford University Press, Oxford1992: 2165-2188Google Scholar.Table 2Comparison of the transport properties of rNaSi-1, mNaSi-1 and hNaSi-1rNaSi-1OocytesElectrophysmNaSi-1 OocyteshNaSi-1 OocytesVmax for sulfate pmol/h43±2—49±4180±9Km for sulfate mmol/L0.62±0.080.09±0.030.20±0.060.31±0.06Vmax for sodium pmol/h17±1—15±125±2Km for sodium mmol/L17±371±1121±224±2Hill coefficient for sulfate1111Hill coefficient for sodium1.8±0.42.8±0.42.8±0.62.6±0.6 Open table in a new tab Recently, we have cloned and characterized the mouse NaSi-1 gene, designated Nas132Beck L. Markovich D. The mouse Na+-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D.J Biol Chem. 2000; 275: 11800-11890Google Scholar. The Nas1 gene is a single copy gene comprising 15 exons and 14 introns spread over 75 kb without obvious pattern of exon organization. The translation initiation site is present in exon 1, and transcription initiation occurs from a single site, 29 bp downstream to a TATA-box-like sequence. Superimposition of the Nas1 exon boundaries on the mNaSi-1 protein secondary structure model shows that splicing mostly occurred near membrane/aqueous transitions, as was also observed with the SGLT1 gene40Turk E. Martin M.G. Wright E.M. Structure of the human Na+/glucose cotransporter gene SGLT1.J Biol Chem. 1994; 269:
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