Na+-sulfate cotransporter SLC13A1.

Sulfate is essential for normal physiology. The kidney plays a major role in sulfate homeostasis. Sulfate is freely filtered and strongly reabsorbed in the proximal tubule. The apical membrane Na(+)-sulfate cotransporter NaS1 (SLC13A1) mediates sulfate (re)absorption across renal proximal tubule and small intestinal epithelia. NaS1 encodes a 595-amino acid (≈ 66 kDa) protein with 13 putative transmembrane domains. Its substrate preferences are sodium and sulfate, thiosulfate, and selenate, and its activity is inhibited by molybdate, selenate, tungstate, thiosulfate, succinate, and citrate. NaS1 is primarily expressed in the kidney (proximal tubule) and intestine (duodenum to colon). NaS1 expression is down-regulated in the renal cortex by high sulfate diet, hypothyroidism, vitamin D depletion, glucocorticoids, hypokalemia, metabolic acidosis, and NSAIDs and up-regulated by low sulfate diet, thyroid hormone, vitamin D supplementation, growth hormone, chronic renal failure, and during post-natal growth. Disruption of murine NaS1 gene leads to hyposulfatemia and hypersulfaturia, as well as changes in metabolism, growth, fecundity, behavior, gut physiology, and liver detoxification. This suggests that NaS1 is an important sulfate transporter and its disruption leads to perturbed sulfate homeostasis, which contributes to numerous pathophysiological conditions.

Regulation of murine hepatic hydroxysteroid sulfotransferase expression in hyposulfatemic mice and in a cell model of 3'-phosphoadenosine-5'-phosphosulfate deficiency.

The cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of nucleophilic substrates, and the cofactor for sulfonation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is biosynthesized from sulfate and ATP. The phenotype of male knockout mice for the NaS1 sodium sulfate cotransporter includes hyposulfatemia and increased hepatic expression of mouse cytoplasmic sulfotransferase Sult2a and Sult3a1. Here we report that in 8-week-old female NaS1-null mice, hepatic Sult2a1 mRNA levels were ∼51-fold higher than they were in a wild-type liver but expression of no other Sult was affected. To address whether hyposulfatemia-inducible Sult2a1 expression might be due to reduced PAPS levels, we stably knocked down PAPS synthases 1 and 2 in HepG2 cells (shPAPSS1/2 cells). When a reporter plasmid containing at least 233 nucleotides (nt) of Sult2a1 5'-flanking sequence was transfected into shPAPSS1/2 cells, reporter activity was significantly increased relative to the activity that was seen for reporters containing 179 or fewer nucleotides. Mutation of an IR0 (inverted repeat of AGGTCA, with 0 intervening bases) nuclear receptor motif at nt -191 to 180 significantly attenuated the PAPSS1/2 knockdown-mediated increase. PAPSS1/2 knockdown significantly activated farnesoid X receptor (FXR), retinoid-related orphan receptor, and pregnane X receptor responsive reporters, and treatment with the FXR agonist GW4064 [3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole] increased Sult2a1 promoter activity when the IR0 was intact. Transfection of shPAPSS1/2 cells with FXR small interfering RNA (siRNA) significantly reduced the Sult2a1 promoter activity. The impact of PAPSS1/2 knockdown on Sult2a1 promoter activity was recapitulated by knocking down endogenous SULT2A1 expression in HepG2 cells. We propose that hyposulfatemia leads to hepatic PAPS depletion, which causes loss of SULT2A1 activity and results in accumulation of nonsulfated bile acids and FXR activation.

Sodium-sulfate/carboxylate cotransporters (SLC13).

The SLC13 gene family is comprised of five sequence related proteins that are found in animals, plants, yeast and bacteria. Proteins encoded by the SLC13 genes are divided into the following two groups of transporters with distinct anion specificities: the Na(+)-sulfate (NaS) cotransporters and the Na(+)-carboxylate (NaC) cotransporters. Members of this gene family (in ascending order) are: SLC13A1 (NaS1), SLC13A2 (NaC1), SLC13A3 (NaC3), SLC13A4 (NaS2) and SLC13A5 (NaC2). SLC13 proteins encode plasma membrane polypeptides with 8-13 putative transmembrane domains, and are expressed in a variety of tissues. They are all Na(+)-coupled symporters with strong cation preference for Na(+), and insensitive to the stilbene 4, 4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Their Na(+):anion coupling ratio is 3:1, indicative of electrogenic properties. They have a substrate preference for divalent anions, which include tetra-oxyanions for the NaS cotransporters or Krebs cycle intermediates (including mono-, di- and tricarboxylates) for the NaC cotransporters. This review will describe the molecular and cellular mechanisms underlying the biochemical, physiological and structural properties of the SLC13 gene family.

Sat1 is dispensable for active oxalate secretion in mouse duodenum.

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.

Slc13a1 and Slc26a1 KO models reveal physiological roles of anion transporters.

Anion transporters NaS1 (SLC13A1) and Sat1 (SLC26A1) mediate sulfate (re)absorption across renal proximal tubule and small intestinal epithelia, thereby regulating blood sulfate levels. Disruption of murine NaS1 and Sat1 genes leads to hyposulfatemia and hypersulfaturia. Sat1-null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. This review will highlight the current pathophysiological features of NaS1- and Sat1-null mice resulting from alterations in circulating sulfate and oxalate anion levels.

Increased lifespan in hyposulfatemic NaS1 null mice.

Sulfate (SO(4)(2-)) plays an important role in mammalian growth and development. In this study, hyposulfatemic NaS1 null (Nas1-/-) mice were used to investigate the consequences of perturbed SO(4)(2-) homeostasis on longevity. Median life spans were increased (by ≈25%) in male and female Nas1-/- mice when compared with Nas1+/+ mice. At 1 yr of age, serum SO(4)(2-) levels remained low in Nas1-/- mice (≈0.16 mM) when compared to Nas1+/+ mice (≈0.96 mM). RT-PCR revealed increased hepatic mRNA levels of Sirt1 (by ≈60%), Cat (by ≈48%), Hdac3 (by ≈22%), Trp53 and Cd55 (by ≈36%) in Nas1-/- mice, genes linked to ageing. Histological analyses of livers from 2 yr old mice revealed neoplasms in >50% of Nas1+/+ mice but not in Nas1-/- mice. This is the first study to report increased lifespan, decreased hepatic tumours and increased hepatic expression of genes linked to ageing in hyposulfatemic Nas1-/- mice, implicating a potential role of SO(4)(2-) in mammalian longevity and cancer.

Physiological roles of renal anion transporters NaS1 and Sat1.

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.

Fetal loss and hyposulfataemia in pregnant NaS1 transporter null mice.

Sulfate is important for growth and development, and is supplied from mother to fetus throughout pregnancy. We used NaS1 sulfate transporter null (Nas1(-/-)) mice to investigate the role of NaS1 in maintaining sulfate homeostasis during pregnancy and to determine the physiological consequences of maternal hyposulfataemia on fetal, placental and postnatal growth. We show that maternal serum (≤0.5 mM), fetal serum (<0.1 mM) and amniotic fluid (≤0.5 mM) sulfate levels were significantly lower in pregnant Nas1(-/-) mice when compared with maternal serum (≍2.0 mM), fetal serum (≍1.5 mM) and amniotic fluid (≍1.7 mM) sulfate levels in pregnant Nas1(+/+) mice. After 12 days of pregnancy, fetal reabsorptions led to markedly reduced (by ≥50%) fetal numbers in Nas1(-/-) mice. Placental labyrinth and spongiotrophoblast layers were increased (by ≍140%) in pregnant Nas1(-/-) mice when compared to pregnant Nas1(+/+) mice. Birth weights of progeny from female Nas1(-/-) mice were increased (by ≍7%) when compared to progeny of Nas1(+/+) mice. These findings show that NaS1 is essential to maintain high maternal and fetal sulfate levels, which is important for maintaining pregnancy, placental development and normal birth weight.

Physiological roles of mammalian sulfate transporters NaS1 and Sat1.

This review summarizes the physiological roles of the renal sulfate transporters NaS1 (Slc13a1) and Sat1 (Slc26a1). NaS1 and Sat1 encode renal anion transporters that mediate proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria and calcium oxalate urolithiasis. Dysregulation of NaS1 and Sat1 leads to hypersulfaturia, hyposulfatemia and liver damage. Loss of Sat1 leads additionally to hyperoxaluria with hyperoxalemia, nephrocalcinosis and calcium oxalate urolithiasis. These data indicate that the renal anion transporters NaS1 and Sat1 are essential for sulfate and oxalate homeostasis, respectively.

Urolithiasis and hepatotoxicity are linked to the anion transporter Sat1 in mice.

Urolithiasis, a condition in which stones are present in the urinary system, including the kidneys and bladder, is a poorly understood yet common disorder worldwide that leads to significant health care costs, morbidity, and work loss. Acetaminophen-induced liver damage is a major cause of death in patients with acute liver failure. Kidney and urinary stones and liver toxicity are disturbances linked to alterations in oxalate and sulfate homeostasis, respectively. The sulfate anion transporter-1 (Sat1; also known as Slc26a1) mediates epithelial transport of oxalate and sulfate, and its localization in the kidney, liver, and intestine suggests that it may play a role in oxalate and sulfate homeostasis. To determine the physiological roles of Sat1, we created Sat1-/- mice by gene disruption. These mice exhibited hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate stones in their renal tubules and bladder. Sat1-/- mice also displayed hypersulfaturia, hyposulfatemia, and enhanced acetaminophen-induced liver toxicity. These data suggest that Sat1 regulates both oxalate and sulfate homeostasis and may be critical to the development of calcium oxalate urolithiasis and hepatotoxicity.

Enhanced tumor growth in the NaS1 sulfate transporter null mouse.

Sulfate plays an important role in maintaining normal structure and function of tissues, and its content is decreased in certain cancers including lung carcinoma. In this study, we investigated tumor growth in a mouse model of hyposulfatemia (Nas1(-/-)) and compared it to wild-type (Nas1(+/+)) mice. Lung epithelial tumor cells (TC-1 cell line) were injected subcutaneously into male Nas1(-/-) and Nas1(+/+) mice on a mixed 129Sv and C57BL/6 genetic background. Tumor sections were stained with anti-glycosaminoglycan antibodies to assess the distribution of proteoglycans and Gomori's trichrome to detect collagen. After 14 days, tumor weights were markedly increased (by approximately 12-fold) in Nas1(-/-) mice when compared with Nas1(+/+) mice. Histological analyses of tumors revealed increased (by approximately 2.4-fold) vessel content, as well as markedly reduced collagen and immunoreactivity against glycosaminoglycan structural epitopes in the tumors from Nas1(-/-) mice. No significant differences were found for the growth of cultured TC-1 cells supplemented with Nas1(-/-) or Nas1(+/+) serum, as determined by (3)H-thymidine incorporation, implying that the cell culture conditions may not reflect the in vivo situation of enhanced tumor growth. This study has revealed increased tumor growth and an altered extracellular tumor matrix in hyposulfatemic Nas1(-/-) mice. These findings highlight the importance of blood sulfate levels as a possible modulator of tumor growth, and could lead to future cancer studies in humans with altered sulfate homeostasis.

Kidney transcriptome reveals altered steroid homeostasis in NaS1 sulfate transporter null mice.

Sulfate is essential for human growth and development, and circulating sulfate levels are maintained by the NaS1 sulfate transporter which is expressed in the kidney. Previously, we generated a NaS1-null (Nas1(-/-)) mouse which exhibits hyposulfatemia. In this study, we investigated the kidney transcriptome of Nas1(-/-) mice. We found increased (n=25) and decreased (n=60) mRNA levels of genes with functional roles that include sulfate transport and steroid metabolism. Corticosteroid-binding globulin was the most up-regulated gene (110% increase) in Nas1(-/-) mouse kidney, whereas the sulfate anion transporter-1 (Sat1) was among the most down-regulated genes (>or=50% decrease). These findings led us to investigate the circulating and urinary steroid levels of Nas1(-/-) and Nas1(+/+) mice, which revealed reduced blood levels of corticosterone ( approximately 50% decrease), dehydroepiandrosterone (DHEA, approximately 30% decrease) and DHEA-sulfate ( approximately 40% decrease), and increased urinary corticosterone ( approximately 16-fold increase) and DHEA ( approximately 40% increase) levels in Nas1(-/-) mice. Our data suggest that NaS1 is essential for maintaining a normal metabolic state in the kidney and that loss of NaS1 function leads to reduced circulating steroid levels and increased urinary steroid excretion.

Functional and structural characterization of the zebrafish Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1).

Sulfate plays an essential role during growth, development and cellular metabolism. In this study, we characterized the function and structure of the zebrafish (Danio rerio) Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1). Zebrafish NaS1 encodes a protein of 583 amino acids with 13 putative transmembrane domains. Expression of zebrafish NaS1 protein in Xenopus oocytes led to Na+-sulfate cotransport, which was significantly inhibited by thiosulfate, selenate, molybdate, and tungstate. Zebrafish NaS1 transport kinetics were: V(max) = 1,731.670 +/- 92.853 pmol sulfate/oocyte.hour and K(m) = 1.414 +/- 0.275 mM for sulfate and V(max) = 307.016 +/- 32.992 pmol sulfate/oocyte x hour, K(m) = 24.582 +/- 4.547 mM and n (Hill coefficient) = 1.624 +/- 0.354 for sodium. Zebrafish NaS1 mRNA is developmentally expressed in embryos from day 1 postfertilization and in the intestine, kidney, brain, and eye of adult zebrafish. The zebrafish NaS1 gene slc13a1 contains 15 exons spanning 8,716 bp. Characterization of the zebrafish NaS1 contributes to a greater understanding of sulfate transporters in a well-defined genetic model and will allow the elucidation of evolutionary and functional relationships among vertebrate sulfate transporters.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Expression cloning and radiotracer uptakes in Xenopus laevis oocytes.

This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.

Hyperserotonaemia and reduced brain serotonin levels in NaS1 sulphate transporter null mice.

Sulphate (SO4) is conjugated to numerous endogenous compounds, including serotonin (5-HT). The NaS1 sulphate transporter is primarily expressed in the kidney, where it maintains blood SO4 concentrations. Previously, we generated NaS1 null (Nas1) mice, which have hyposulphataemia and decreased anxiety and locomotor activity. In this study, we investigated 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentrations, and 5-HT receptor mRNA levels. Nas1 mice exhibited a doubling in blood 5-HT concentration, but a 12% reduction in brain levels of 5-HT and 5-hydroxyindole acetic acid. Brain 5-HT1A and 5-HT1B receptor mRNA levels were increased by 50%, compared with wild-type mice. Our data indicate that decreased circulating SO4 concentrations modulate 5-HT neurotransmitter and receptor levels, in a manner consistent with the behavioural phenotypes of Nas1 mice.

Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines.

NaSi-1 encodes a Na(+)-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.

Specificity and regulation of renal sulfate transporters.

Sulfate is essential for normal cellular function. The kidney plays a major role in sulfate homeostasis. Sulfate is freely filtered and then undergoes net reabsorption in the proximal tubule. The apical membrane Na(+)/sulfate cotransporter NaS1 (SLC13A1) has a major role in mediating proximal tubule sulfate reabsorption, as demonstrated by the findings of hyposulfatemia and hypersulfaturia in Nas1-null mice. The anion exchanger SAT1 (SLC26A1), the founding member of the SLC26 sulfate transporter family, mediates sulfate exit across the basolateral membrane to complete the process of transtubular sulfate reabsorption. Another member of this family, CFEX (SLC26A6), is present at the apical membrane of proximal tubular cells. It also can transport sulfate by anion exchange, which probably mediates backflux of sulfate into the lumen. Knockout mouse studies have demonstrated a major role of CFEX as an apical membrane Cl(-)/oxalate exchanger that contributes to NaCl reabsorption in the proximal tubule. Several additional SLC26 family members mediate sulfate transport and show some level of renal expression (e.g., SLC26A2, SLC26A7, SLC26A11). Their roles in mediating renal tubular sulfate transport are presently unknown. This paper reviews current data available on the function and regulation of three sulfate transporters (NaS1, SAT1, and CFEX) and their physiological roles in the kidney.

Sulfate transport by SLC26 transporters.

Sulfate is the fourth most abundant anion in human plasma that is essential for numerous physiological functions, including biotransformation of xenobiotics, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), adrenergic stimulants/blockers and analgesics. Sulfate is also required for activation of many endogenous compounds (heparin, heparan sulfate, dermatan sulfate, bile acids) and utilized in the metabolism of neurotransmitters. Sulfation of structural components, including glycosaminoglycans and cerebroside sulfate, is essential for the maintenance of normal structure and function of tissues. Due to its hydrophilic nature, sulfate cannot readily cross the lipid bilayer of cells, thus plasma membrane proteins, known as sulfate transporters, are required for the movement of sulfate into/out of cells. Sulfate transporters can be divided into two distinct groups: Na+-dependent sulfate transporters belonging to the SLC13 gene family and Na+-independent sulfate transporters (antiporters, exchangers) belonging to SLC26 gene family. There are 11 members of the SLC26 family (including Sat1, DTDST, CLD, pendrin, prestin, cfex) whose structures and functions have been only partially characterized. In this presentation, the current information on the structures and functions of the sulfate transporters in the SLC26 gene family will be described and the issue that certain members of this family are unable to transport sulfate, will be addressed.

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): Structure and expression.

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na(+)-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6B1 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.