Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).     

SLC2A9 is a high-capacity urate transporter in humans

Background

Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man.

Methods and Findings

We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K _i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82).

Conclusions

This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.     

Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.     

Mutations in Glucose Transporter 9 Gene SLC2A9 Cause Renal Hypouricemia

Renal hypouricemia is an inherited disorder characterized by impaired renal urate (uric acid) reabsorption and subsequent low serum urate levels, with severe complications such as exercise-induced acute renal failure and nephrolithiasis. We previously identified SLC22A12, also known as URAT1, as a causative gene of renal hypouricemia. However, hypouricemic patients without URAT1 mutations, as well as genome-wide association studies between urate and SLC2A9 (also called GLUT9), imply that GLUT9 could be another causative gene of renal hypouricemia. With a large human database, we identified two loss-of-function heterozygous mutations in GLUT9, which occur in the highly conserved “sugar transport proteins signatures 1/2.” Both mutations result in loss of positive charges, one of which is reported to be an important membrane topology determinant. The oocyte expression study revealed that both GLUT9 isoforms showed high urate transport activities, whereas the mutated GLUT9 isoforms markedly reduced them. Our findings, together with previous reports on GLUT9 localization, suggest that these GLUT9 mutations cause renal hypouricemia by their decreased urate reabsorption on both sides of the renal proximal tubules. These findings also enable us to propose a physiological model of the renal urate reabsorption in which GLUT9 regulates serum urate levels in humans and can be a promising therapeutic target for gout and related cardiovascular diseases.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

Increased expression of SLC2A9 decreases urate excretion from the kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

SLC41A2 encodes a plasma-membrane Mg2+ transporter

The TRPM7 (transient receptor potential melastatin 7) ion channel has been implicated in the uptake of Mg2+ into vertebrate cells, as elimination of TRPM7 expression through gene targeting in DT40 B-lymphocytes renders them unable to grow in the absence of supplemental Mg2+. However, a residual capacity of TRPM7-deficient cells to accumulate Mg2+ and proliferate when provided with supplemental Mg2+ suggests the existence of Mg2+ uptake mechanism(s) other than TRPM7. Evaluation of the expression of several members of the SLC41 (solute carrier family 41) family, which exhibit homology with the MgtE class of prokaryotic putative bivalent-cation transporters, demonstrated that one, SLC41A2 (solute carrier family 41 member 2), is expressed in both wild-type and TRPM7-deficient DT40 cells. Characterization of heterologously expressed SLC41A2 protein indicated that it is a plasma-membrane protein with an N-terminus-outside/C-terminus-inside 11-TM (transmembrane)-span topology, consistent with its functioning as a trans-plasma-membrane transporter. In contrast with a previous report of ion-channel activity associated with SLC41A2 expression in oocytes, investigation of whole cell currents in SLC41A2-expressing DT40 cells revealed no novel currents of any type associated with SLC41A2 expression. However, expression of SLC41A2 in TRPM7-deficient cells under the control of a doxycycline-inducible promoter was able to conditionally enhance their net uptake of 26Mg2+ and conditionally and dose-dependently provide them with the capacity to grow in the absence of supplemental Mg2+, observations strongly supporting a model whereby SLC41A2 directly mediates trans-plasma-membrane Mg2+ transport. Overall, our results suggest that SLC41A2 functions as a plasma-membrane Mg2+ transporter in vertebrate cells.     

SLC41A1 is a novel mammalian Mg2+ carrier

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments. Nevertheless, in a strain of Salmonella enterica exhibiting disruption of all three distinct magnesium transport systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally substitutes these transporters and restores the growth of the mutant bacteria at magnesium concentrations otherwise non-permissive for growth. Thus, we have identified human SLC41A1 as being a bona fide magnesium transporter. Most importantly, overexpressed SLC41A1 provide HEK293 cells with an increased magnesium efflux capacity. With outwardly directed Mg(2+) gradients, a SLC41A1-dependent reduction of the free intracellular magnesium concentration accompanied by a significant net decrease of the total cellular magnesium concentration could be observed in such cells. SLC41A1 activity is temperature-sensitive but not sensitive to the only known magnesium channel blocker, cobalt(III) hexaammine. Taken together, these data functionally identify SLC41A1 as a mammalian carrier mediating magnesium efflux.     

Facilitated diffusion of urate in avian brush-border membrane vesicles

Membrane transport pathways mediatingtranscellular secretion of urate across the proximal tubule wereinvestigated in brush-border membrane vesicles (BBMV) isolated fromavian kidney. An inside-positive K diffusion potential induced aconductive uptake of urate to levels exceeding equilibrium.Protonophore-induced dissipation of membrane potential significantlyreduced voltage-driven urate uptake. Conductive uptake of urate wasinhibitor sensitive, substrate specific, and a saturable function ofurate concentration. Urate uptake was trans-stimulated byurate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate.Urate uptake was unaffected by an outward -ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in thepresence and absence of an imposed inside-alkaline pH gradient or anoutward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux acrossthe brush border membrane of urate-secreting proximal tubule cells.

     

Complex Analysis of Urate Transporters SLC2A9, SLC22A12 and Functional Characterization of Non-Synonymous Allelic Variants of GLUT9 in the Czech Population: No Evidence of Effect on Hyperuricemia and Gout

Objective

Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.

Methods

The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.

Results

We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.

Conclusion

Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.     

Insights into the transport side of the human SLC38A9 transceptor

The lysosomal amino acid transporter SLC38A9 is referred to as transceptor, i.e. a transporter with a receptor function. The protein is responsible for coupling amino acid transport across the lysosomal membrane according to the substrate availability to mTORC1 signal transduction. This process allows cells to sense amino acid level responding to growth stimuli in physiological and pathological conditions triggering mTOR regulation. The main substrates underlying this function are glutamine and arginine. The functional and kinetic characterization of glutamine and arginine transport was performed using human SLC38A9 produced in E. coli, purified by affinity chromatography and reconstituted in liposomes. A cooperative behaviour for the wild type protein was revealed for both the substrates. A novel Na⁺ binding site, namely T453, was described by combined approaches of bioinformatics, site-directed mutagenesis and transport assay. Stimulation by cholesterol of glutamine and arginine transport was observed. The biological function of SLC38A9 relies on the interaction between its N-terminus and components of the mTOR complex; a deletion mutant of the N-terminus tail was produced and transport of glutamine was assayed revealing that this portion does not play any role in the intrinsic transport function of the human SLC38A9. Different features for glutamine and arginine transport were revealed: human SLC38A9 is competent for glutamine efflux, while that of arginine is negligible. In line with these results, imposed ?pH stimulated glutamine, not arginine transport. Arginine plays, on the contrary, a modulatory function and is able to stimulate glutamine efflux. Interestingly, reciprocal inhibition experiments also supported by bioinformatics, suggested that glutamine and arginine may bind to different sites in the human SLC38A9 transporter.     

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.     

Mutations in the SLC2A9 gene cause hyperuricosuria and hyperuricemia in the dog

Allantoin is the end product of purine catabolism in all mammals except humans, great apes, and one breed of dog, the Dalmatian. Humans and Dalmatian dogs produce uric acid during purine degradation, which leads to elevated levels of uric acid in blood and urine and can result in significant diseases in both species. The defect in Dalmatians results from inefficient transport of uric acid in both the liver and renal proximal tubules. Hyperuricosuria and hyperuricemia (huu) is a simple autosomal recessive trait for which all Dalmatian dogs are homozygous. Therefore, in order to map the locus, an interbreed backcross was used. Linkage mapping localized the huu trait to CFA03, which excluded the obvious urate transporter 1 gene, SLC22A12. Positional cloning placed the locus in a minimal interval of 2.5 Mb with a LOD score of 17.45. A critical interval of 333 kb containing only four genes was homozygous in all Dalmatians. Sequence and expression analyses of the SLC2A9 gene indicated three possible mutations, a missense mutation (G616T;C188F) and two promoter mutations that together appear to reduce the expression levels of one of the isoforms. The missense mutation is associated with hyperuricosuria in the Dalmatian, while the promoter SNPs occur in other unaffected breeds of dog. Verification of the causative nature of these changes was obtained when hyperuricosuric dogs from several other breeds were found to possess the same combination of mutations as found in the Dalmatian. The Dalmatian dog model of hyperuricosuria and hyperuricemia underscores the importance of SLC2A9 for uric acid transport in mammals.     

Downregulation of the Creatine Transporter SLC6A8 by JAK2

Janus-activated kinase-2 (JAK2) participates in the regulation of the Na⁺-coupled glucose transporter SGLT1 and the Na⁺-coupled amino acid transporter SLC6A19. Concentrative cellular creatine uptake is similarly accomplished by Na⁺-coupled transport. The carrier involved is SLC6A8 (CreaT). The present study thus explored whether JAK2 regulates the activity of SLC6A8. To this end, cRNA encoding SLC6A8 was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2. Electrogenic creatine transport was determined in those oocytes by dual-electrode voltage-clamp experiments. In oocytes injected with cRNA encoding SLC6A8 but not in oocytes injected with water or with cRNA encoding JAK2 alone, addition of 1 mM creatine to the extracellular bath generated an inward current (I _crea). In SLC6A8 expressing oocytes I _crea was significantly decreased by coexpression of JAK2 or ^V617FJAK2 but not by coexpression of ^K882EJAK2. According to kinetic analysis, coexpression of JAK2 decreased the maximal transport rate without significantly modifying the affinity of the carrier. In oocytes expressing SLC6A8 and ^V617FJAK2 I _crea was gradually increased by the JAK2 inhibitor AG490 (40 μM). In SLC6A8 and JAK2 coexpressing oocytes the decline of I _crea following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of JAK2. In conclusion, JAK2 is a novel regulator of the creatine transporter SLC6A8, which downregulates the carrier, presumably by interference with carrier protein insertion into the cell membrane.     

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.     

Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na⁺-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na⁺-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na⁺-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36 ± 0.04 mM. Na⁺-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8 ± 0.4, indicating involvement of more than one Na⁺ in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na⁺-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19 ± 0.01 mM. The Na⁺-activation kinetics is sigmoidal with a Hill coefficient of 2.3 ± 0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14 ± 1 μM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na⁺-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na⁺ gradient-driven pyroglutamate uptake was stimulated by an inside-negative K⁺ diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.     

Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

Background

Many genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.

Methodology/Principal Findings

Isoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.

Conclusions/Significance

SLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.     

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

The H(+)-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly-Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly-Sar uptake through a reduction in capacity (V(max)) without any effect on affinity (K(m)). The reduction in dipeptide transport was dependent upon both extracellular Na(+) and apical pH but was not observed in the presence of the selective Na(+)/H(+) exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H(+)-electrochemical gradient which, in turn, acts as the driving force for H(+)-coupled solute transport. Uptake of beta-alanine, a substrate for the H(+)-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H(+)-electrochemical gradient and may, therefore, be a site for both nutrient-drug and drug-drug interactions in the small intestine.