Rare SLC13A1 variants associate with intervertebral disc disorder highlighting role of sulfate in disc pathology.

Back pain is a common and debilitating disorder with largely unknown underlying biology. Here we report a genome-wide association study of back pain using diagnoses assigned in clinical practice; dorsalgia (119,100 cases, 909,847 controls) and intervertebral disc disorder (IDD) (58,854 cases, 922,958 controls). We identify 41 variants at 33 loci. The most significant association (ORIDD = 0.92, P = 1.6 × 10-39; ORdorsalgia = 0.92, P = 7.2 × 10-15) is with a 3'UTR variant (rs1871452-T) in CHST3, encoding a sulfotransferase enzyme expressed in intervertebral discs. The largest effects on IDD are conferred by rare (MAF = 0.07 - 0.32%) loss-of-function (LoF) variants in SLC13A1, encoding a sodium-sulfate co-transporter (LoF burden OR = 1.44, P = 3.1 × 10-11); variants that also associate with reduced serum sulfate. Genes implicated by this study are involved in cartilage and bone biology, as well as neurological and inflammatory processes.

Thermostability-based binding assays reveal complex interplay of cation, substrate and lipid binding in the bacterial DASS transporter, VcINDY.

The divalent anion sodium symporter (DASS) family of transporters (SLC13 family in humans) are key regulators of metabolic homeostasis, disruption of which results in protection from diabetes and obesity, and inhibition of liver cancer cell proliferation. Thus, DASS transporter inhibitors are attractive targets in the treatment of chronic, age-related metabolic diseases. The characterisation of several DASS transporters has revealed variation in the substrate selectivity and flexibility in the coupling ion used to power transport. Here, using the model DASS co-transporter, VcINDY from Vibrio cholerae, we have examined the interplay of the three major interactions that occur during transport: the coupling ion, the substrate, and the lipid environment. Using a series of high-throughput thermostability-based interaction assays, we have shown that substrate binding is Na+-dependent; a requirement that is orchestrated through a combination of electrostatic attraction and Na+-induced priming of the binding site architecture. We have identified novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carboxylate groups in the ligand that are precisely distanced to satisfy carboxylate interaction regions of the substrate-binding site. We have also identified a complex relationship between substrate and lipid interactions, which suggests a dynamic, regulatory role for lipids in VcINDY's transport cycle.

From Genotype to Phenotype: Nonsense Variants in SLC13A1 Are Associated with Decreased Serum Sulfate and Increased Serum Aminotransferases.

Using genomic applications to glean insights into human biology, we systematically searched for nonsense single nucleotide variants (SNVs) that are rare in the general population but enriched in the Old Order Amish (Amish) due to founder effect. We identified two nonlinked, nonsense SNVs (R12X and W48X) in SLC13A1 (allele frequencies 0.29% and 0.74% in the Amish; enriched 1.2-fold and 3.7-fold, compared to the outbred Caucasian population, respectively). SLC13A1 encodes the apical sodium-sulfate cotransporter (NaS1) responsible for sulfate (re)absorption in the kidneys and intestine. SLC13A1 R12X and W48X were independently associated with a 27.6% (P = 2.7 × 10(-8)) and 27.3% (P = 6.9 × 10(-14)) decrease in serum sulfate, respectively (P = 8.8 × 10(-20) for carriers of either SLC13A1 nonsense SNV). We further performed the first exome- and genome-wide association study (ExWAS/GWAS) of serum sulfate and identified a missense variant (L348P) in SLC26A1, which encodes the basolateral sulfate-anion transporter (Sat1), that was associated with decreased serum sulfate (P = 4.4 × 10(-12)). Consistent with sulfate's role in xenobiotic detoxification and protection against acetaminophen-induced hepatotoxicity, SLC13A1 nonsense SNV carriers had higher aminotransferase levels compared to noncarriers. Furthermore, SLC26A1 L348P was associated with lower whole-body bone mineral density (BMD) and higher serum calcium, consistent with the osteochondrodysplasia exhibited by dogs and sheep with naturally occurring, homozygous, loss-of-function mutations in Slc13a1 This study demonstrates the power and translational potential of systematic identification and characterization of rare, loss-of-function variants and warrants additional studies to better understand the importance of sulfate in human physiology, disease, and drug toxicity.

Determination of Unbound Partition Coefficient and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake.

Unbound partition coefficient (Kpuu) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transporter-transfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher Kpuu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (Km) of SLC13A5 was estimated to be 24 µM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

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.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters.

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

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.

In a shake of a lamb's tail: using genomics to unravel a cause of chondrodysplasia in Texel sheep.

Chondrodysplasia in Texel sheep is a recessively inherited disorder characterized by dwarfism and angular deformities of the forelimbs. A genome-wide association study using the Illumina OvineSNP50 BeadChip on 15 sheep diagnosed as affected and eight carriers descended from three affected rams was conducted to uncover the genetic cause. A homozygous region of 25 consecutive single nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 1 Mbp on ovine chromosome 4. Seven positional candidate genes - including the solute carrier family 13 (sodium/sulphate symporters), member 1 (SLC13A1) - were identified and used to search for new SNPs for fine mapping of the causal locus. The SLC13A1 gene, encoding a sodium/sulphate transporter, was the primary candidate gene attributable to similar phenotypes observed in the Slc13a1 knockout mouse model. We discovered a 1-bp deletion of T (g.25513delT) at the 107 bp position of exon 3 in the SLC13A1 gene. Genotyping by direct sequencing and restriction fragment length polymorphism analysis for this mutation showed that all 15 affected sheep were g.25513delT/g.25513delT; the eight carriers were g.25513delT/T and 54 normal controls were T/T. The mutation g.25513delT shifts the open reading frame of SLC13A1 to introduce a stop codon and truncate C-terminal amino acids. It was concluded that the g.25513delT mutation in the SLC13A1 gene was responsible for the chondrodysplasia seen in these Texel sheep. This knowledge can be used to identify carriers with the defective g.[25513delT] allele to avoid at-risk matings to improve animal welfare and decrease economic losses.

Placental sulphate transport: a review of functional and molecular studies.

Sulphate is required by the feto-placental unit for a number of important conjugation and biosynthetic pathways. Functional studies performed several decades ago established that sulphate transport in human placental microvillus and basal membrane vesicles was mainly via a DIDS-sensitive anion-exchange mechanism. In contrast, no evidence was found for Na⁺-dependent transport. Studies performed using isolated human placental tissue confirmed anion-exchange as the main mechanism. More recently, molecular studies have established the presence of anion-exchange proteins which could play a role in transplacental sulphate movement. However, the presence of transcripts for NaS2 has been reported and has prompted the suggestion that Na⁺-sulphate cotransport may play an important role in maternal-fetal sulphate transport. This article reviews our present knowledge of placental sulphate transport, both functional and molecular, and attempts to form a model based on the available evidence.

Partial deletion of the sulfate transporter SLC13A1 is associated with an osteochondrodysplasia in the Miniature Poodle breed.

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8:8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (P(raw) <10(-8)). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.

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.

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.

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.