OATP1B1 Research Articles

SLC22A12 W258X frequency according to serum uric acid level among Japanese health checkup examinees.

Although the SLC22A12 (uric acid transporter 1) 258X allele is known to cause hypouricemia, the genotype frequency according to the serum uric acid (SUA) level has not been reported. This study investigated the SLC22A12 258WX frequency according to SUA levels among Japanese health-checkup examinees. In addition, the changes were reported in SUA levels during five years for individuals with 258WX. Subjects were 746 Japanese aged 39-86 years in 2003. Their SUA records were linked during the five years from 2003 to 2007. SLC22A12 W258X was genotyped using a polymerase chain reaction with confronting two-pair primers. The 258X allele comprised 1.9% (95% CI, 1.3-2.8%) of all the subjects. Among those with SUA <3.0 mg/dL, 258WX was more common in males (66.7%, 95% CI, 22.2-95.7%) than in females (39.3%, 95% CI, 21.5-59.4%). Among subjects with a SUA of 3.0-4.9 mg/dL, those with 258WX totaled 10.7% (95% CI, 4.0-21.9%) and 2.6% (95% CI, 1.1-5.0%), respectively. There were no subjects with 258WX among those with a SUA of 5.0 mg/dL or more. During the five years from 2003 to 2007, the changes in SUA among 23 individuals with 258WX were found to be similar to those among 258WW subjects (n=536). This study indicated that SLC22A12 258WX was more common among those with a lower serum uric acid concentration. The observed SUA level changes in individuals with 258WX suggested that lifestyle factors could influence the levels of those with 258WX.

Genetic Analysis of Human Urate Transporter 1 (hURAT1) and Glucose Transporter 9 (GLUT9) in Taiwanese Patients with Idiopathic Renal Hypouricemia

Background: The phenotypic characteristics of idiopathic renal hypouricemia (IRH) include impaired renal uric acid re-absorption, low serum uric acid levels, and severe complications such as exercise-induced acute renal failure (EIARF) and nephrolithiasis. IRH is increasingly reported to be associated with inactivating mutations in either SLC22A12 gene encoding the human urate acid transporter 1 (hURAT1) or 5LC2A9 gene encoding glucose transporter 9 (GLUT9) in proximal tubules. Mutations in hURAT1 or GLUT9 have not been reported in Taiwanese patients with IRH. Purpose: To investigate the phenotypic and genetic characteristics in Taiwanese patients with IRH. Patients: Six Taiwanese patients (5 males and 1 female, mean age 30±23 years) with hypouricemia (serum uric acid concentration＜2.3 mg/dL) and concomitant excessive renal uric acid excretion (FE(subscript UA)＞10%) from five unrelated families were enrolled. Their clinical symptoms and biochemical studies were recorded. Methods: Molecular analysis of both SLC22A12 and 5LC2A9 genes was performed by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and direct sequencing. Results: Serum uric acid concentration was 1.16±0.23 mg/dl with a fractional excretion of uric acid of 61±12%. Two male patients experienced exercise-induced acute renal failure requiring hemodialysis. None of them had nephrocalcinosis or uric acid stone. Despite a negative mutation in 5LC2J49, four novel SLC22A12 mutations (W120R, M2 15L, T2 17M and T467M) and one recurrent homozygous (R90H) mutations were identified in five patients. Conclusion: EIARF can develop in Taiwanese patients with IRH, and most of them are males. Mutations in 5LC22A 12 rather than 5LC2A9 cause IRH in patients with FE(subscript UA)＜100%. Other candidate genes need to be identified for IRH patients without 5LC22J4 12 and SLC2A9 gene mutations.

Pharmacogenetics of membrane transporters: an update on current approaches.

This review provides an overview of the pharmacogenetics of membrane transporters including selected ABC transporters (ABCB1, ABCC1, ABCC2, and ABCG2) and OATPs (OATP1B1 and OATP1B3). Membrane transporters are heavily involved in drug clearance and alters drug disposition by actively transporting substrate drugs between organs and tissues. As such, polymorphisms in the genes encoding these proteins may have significant effects on the absorption, distribution, metabolism and excretion of compounds, and may alter pharmacodynamics of many agents. This review discusses the techniques used to identify substrates and inhibitors of these proteins and subsequently to assess the effect of genetic mutation on transport, both in vitro and in vivo. A comprehensive list of substrates for the major drug transporters is included. Finally, studies linking transporter genotype with clinical outcomes are discussed.

A Variant of the SLC10A2 Gene Encoding the Apical Sodium-Dependent Bile Acid Transporter Is a Risk Factor for Gallstone Disease

BackgroundCholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated. As one of these mechanisms, a decreased expression of the ileal apical sodium-dependent bile acid transporter gene SLC10A2 in gallstone carriers was described previously. In this study the SLC10A2 gene was investigated to identify novel genetic variants and their association with gallstone formation.Methodology/Principal FindingsStudy subjects were selected with the presence or absence of gallstones confirmed by ultrasound and medical history. Genomic DNA was obtained from blood leukocytes. Sequence analysis was performed of all six exonic and flanking regions as well as of 2,400 base pairs of the SLC10A2 promoter in a cohort of gallstone carriers and control subjects from Stuttgart, Germany. Genotype frequencies of newly identified genetic variants (n = 6) and known single nucleotide polymorphisms (n = 24) were established using MALDI-TOF mass spectrometry. Six new genetic variants were found within the SLC10A2 gene. Although none of the variants was linked to gallstone disease in the Stuttgart cohort overall, the minor allele of SNP rs9514089 was more prevalent in male non-obese gallstone carriers (p = 0.06680, OR = 11.00). In a separate population from Aachen, Germany, the occurrence of rs9514089 was two-fold higher in gallstone patients (22%) than in corresponding controls (11%) (p = 0.00995, OR = 2.19). In the pooled Aachen/Stuttgart cohort rs9514089 was highly significantly linked to cholelithiasis (p = 0.00767, OR = 2.04). A more frequent occurrence was observed for male gallstone carriers (22%) compared to controls (9%) (p = 0.01017, OR = 2.99), for the total normal weight group (p = 0.00754, OR = 2.90), and for male non-obese gallstone patients (p = 0.01410, OR = 6.85). Moreover, for the minor allele of rs9514089 an association with low plasma cholesterol levels was found especially in gallstone carriers (p = 0.05).Conclusions/SignificanceWe have identified SLC10A2 as a novel susceptibility gene for cholelithiasis in humans. Comprehensive statistical analysis provides strong evidence that rs9514089 is a genetic determinant especially in male non-obese gallstone carriers. The minor allele of rs9514089 is related to differences in plasma cholesterol levels among the subjects.

The modern management of gout

Gout is an inflammatory arthritis characterized by self-limiting but excruciatingly painful acute attacks. These are a consequence of monosodium urate (MSU) crystals being deposited within articular or periarticular tissue. Chronic tophaceous gout can develop after years of acute intermittent gout. Recent discoveries, including the role of the inflammasome and intracellular events demonstrating that pro-inflammatory cytokines, IL-1 beta, -8 and TNF-alpha, promote neutrophil influx. Also, genetic advances with the identification of the URAT-1 transporter and genetic variation in SLC 2A9 as a key regulator of urate homoeostasis, have given us deeper understanding of the pathophysiology of gout, and also allow for more targeted treatments. Hopefully, new and emerging therapeutic options will reduce treatment-resistant gout in patients who are unresponsive or unable to take traditional urate lowering therapy. The development of new therapies may also increase patient numbers being treated in the specialist setting, which may have several secondary benefits.

Déficit homozygote en antithrombine de type II (Leu99Phe) : une cause rare de thromboses artérielles récidivantes

In human, vitamin C (l-ascorbic acid) is an essential micronutrient required for an array of biological functions including enzymatic reactions and antioxidation. We describe here the molecular cloning of a novel human cDNA encoding a vitamin C transporter SVCT1. SVCT1 is largely confined to bulk-transporting epithelia (e.g., kidney and small intestine) with a putative alternative-splice product present in thymus. Applying radiotracer and voltage-clamp approaches in cRNA-injected Xenopus oocytes, we found that SVCT1 mediates saturable, concentrative, high-affinity l-ascorbic acid transport (K0.5 = 50–100 μM) that is electrogenic and can be inhibited by phloretin. SVCT1 displays exquisite substrate selectivity, greatly favoring l-ascorbic acid over its isomers d-isoascorbic acid and dehydroascorbic acid and 2- or 6-substituted analogues, whereas glucose and nucleobases are excluded. We have mapped the SLC23A2 gene (coding for SVCT1) to human chromosome 5 in band 5q31.2-31.3, within a region commonly deleted in malignant myeloid (leukemia) diseases. In addition, we have demonstrated that the human SLC23A1 gene product is a related high-affinity l-ascorbic acid transporter (SVCT2) that is widely distributed in brain, retina, and a host of endocrine and neuroendocrine tissues. The molecular identification of the human l-ascorbic acid transporters now provides the tools with which to investigate their roles in vitamin C metabolism in health and disease.

Association of polymorphisms of solute carrier family 22 member 4/5 genes with Crhon's disease in Chinese Han nationality

Objective To study the association of single nucleotide polymorphisms (SNPs) of solute carrier family 22 member 4 (SLC22A4) and SLC22A5 genes with Crhon's disease (CD) in Chinese Han nationality. Methods SNPs in the entire coding region of SLC22A4 and SLC22A5 genes were screened by direct DNA sequencing in 80 CD patients and 80 healthy subjects, and statistical in Han population. Five SNPs were found in entire coding region (2 in SLC22A4 gene and 3 in distribution of the alleles and genotypes of SLC22A4 and SLC22A5 polymorphisms between CD patients and healthy controls. Conclusion There is no correlation of SLC22A4 and SLC22A5 with CD in Chinese Han nationality. Key words: Crhon's disease; Single nucleotide polymorphism; Case-control study

Effects of common haplotypes of the ileal sodium dependent bile acid transporter gene on the development of sporadic and familial colorectal cancer: A case control study

BackgroundThe genetics of sporadic and non-syndromic familial colorectal cancer (CRC) is not well defined. However, genetic factors that promote the development of precursor lesions, i.e. adenomas, might also predispose to CRC. Recently, an association of colorectal adenoma with two variants (c.507C>T;p.L169L and c.511G>T;p.A171S) of the ileal sodium dependent bile acid transporter gene (SLC10A2) has been reported. Here, we reconstructed haplotypes of the SLC10A2 gene locus and tested for association with non-syndromic familial and sporadic CRC compared to 'hyper-normal' controls who displayed no colorectal polyps on screening colonoscopy.MethodsWe included 150 patients with sporadic CRC, 93 patients with familial CRC but exclusion of familial adenomatous polyposis and Lynch's syndrome, and 204 'hyper-normal' controls. Haplotype-tagging SLC10A2 gene variants were identified in the Hapmap database and genotyped using PCR-based 5' exonuclease assays with fluorescent dye-labelled probes. Haplotypes were reconstructed using the PHASE algorithm. Association testing was performed with both SNPs and reconstructed haplotypes.ResultsMinor allele frequencies of all SLC10A2 polymorphisms are within previously reported ranges, and no deviations from Hardy-Weinberg equilibrium are observed. However, we found no association with any of the SLC10A2 haplotypes with sporadic or familial CRC in our samples (all P values > 0.05).ConclusionCommon variants of the SLC10A2 gene are not associated with sporadic or familial CRC. Hence, albeit this gene might be associated with early stages of colorectal neoplasia, it appears not to represent a major risk factor for progression to CRC.

Subcellular localization of transporters along the rat blood–brain barrier and blood–cerebral-spinal fluid barrier by in vivo biotinylation

Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood–brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal–lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal–lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood–brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Functional Characterization of Polymorphic Variants in the Human Apical Sodium‐dependent Bile Acid Transporter (ASBT; SLC10A2)

The apical sodium‐dependent bile acid transporter (ASBT) mediates the uptake of bile acids into intestinal enterocytes, supporting a critical role in normal bile acid homeostasis and maintenance of enterohepatic recirculation. Complete loss of function mutations in ASBT are associated with primary bile acid malaborption, characterized by diarrhea, steatorrhea, interruption of bile acid enterohepatic circulation, and reduced plasma cholesterol levels. We identified 6 nonsynonymous polymorphic variants within the exonic regions of the SLC10A2 gene from DNA derived from a large number of ethnically diverse healthy subjects. In general, variants tended to be infrequent and ethnic‐dependent; only one variant, 511GT (Ala171Ser), was found with a relatively common allele frequency (6.0%), in European‐Americans. A recombinant vaccinia‐based system was utilized to heterologously express wild‐type and variant ASBT variants in HeLa cells for functional studies using radiolabeled taurocholate as a substrate. Two variants, 292GA (Val98Ile) and 431GA (Cys144Tyr), were associated with significantly impaired taurocholate transport activity in vitro compared to wild‐type ASBT. Accordingly, polymorphisms in SLC10A2 may have important functional implications to normal bile acid homeostasis and enterohepatic recirculation. This work was supported in part by NIH grants GM54724 (RBK) and GM81363 (RHH).

Mutant of OATP-C positive ion lysine-49 impairs uptaking ability of its substrate

目的 研究人肝脏特异的有机阴离子转运蛋白基因OATP-C分子膜外区保守的49位阳离子赖氨酸,对其有机阴离子底物摄取能力的影响.方法 从人肝脏mRNA中克隆OATP-C野生型基因全长cDNA序列,通过定点突变将高度保守的第49位赖氨酸残基突变为中性氨基酸-苏氨酸,构建其C端的GFP融合蛋白的真核表达载体,转染HEK293细胞,观察突变体的细胞膜定位能力及底物摄取功能的变化.结果 OATP-C野生型及突变体基因均能定位表达于HEK293细胞质膜;[3H]硫酸盐雌酮底物摄取实验显示,突变体5 rain底物摄取量较野生型下降74.18%,浓度依赖的[3H]硫酸盐雌酮底物摄取动力学分析表明,突变体Km增高和vmax降低.结论 OATP-C蛋白膜外区第49位赖氨酸是其关键的功能氨基酸,是其重要的功能结构单位。

Genetic polymorphisms of the urea transporter gene are associated with antihypertensive response to nifedipine GITS

Nifedipine GITS has diuretic and natriuretic properties, which may enhance its antihypertensive efficacy. We assessed contributions of polymorphisms in the urea transporter-A gene (SLC14A2) to interindividual variations in blood pressure (BP) response to nifedipine treatment. 405 subjects from a single Chinese county received a single oral dose of 30 mg nifedipine GITS (gastrointestinal therapeutic system) daily for 16 days. We genotyped two SNPs in SLC14A2 and found significant associations for the Val227Ile (rs1123617) and Ala357Thr (rs3745009) polymorphisms with BP response to nifedipine treatment. After treatment, subjects with either Ala357/Thr357 or Thr357/Thr357 genotypes had significantly smaller mean changes in systolic BP (SBP) (beta +/- SE = -2.87 +/- 1.24 mmHg, p = 0.020) and diastolic BP (DBP) (beta +/- SE = -1.69 +/- 0.62 mmHg, p = 0.006) compared to those with the Ala357/Ala357 genotype. Subjects with either Val227/Ile227 or Ile227/Ile227 genotypes had significantly larger mean changes in SBP (beta +/- SE = 3.13 +/- 1.19, p = 0.009) and DBP (beta +/- SE = 1.50 +/- 0.60 mmHg, p = 0.013) compared with those with the Val227/Val227 genotype after treatment. Subjects carrying both the Ala357/Ala357 genotype in the Ala357Thr polymorphism and either Val227/Ile227 or Ile227/Ile227 genotypes in the Val227Ile polymorphism had the highest mean change in SBP and DBP. Our study supports the conclusion that polymorphisms in the SLC14A2 gene can predict the antihypertensive efficacy of nifedipine GITS.

The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver

Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug–drug and drug–endogenous substance interactions.

Bile acid (BA) metabolism is impaired in mice with defective peroxisomal BA synthesis

Impairment of peroxisomal BA synthesis is a characteristic feature of Scp2 (-/-) mice. This study investigated how this may impact BA metabolism. In separate groups of male mice (n=6–9) lipid analysis of hepatic and gallbladder bile was performed. Livers and sequential intestinal fractions were harvested to isolate total RNA, homogenate and plasma membranes. Steady-state gene expression of basolateral (Slc10a1, Slc21a1) and canalicular (Abcb4, Abcb11, Abcc2) transporters was studied and correlated with serum and biliary lipids. Size and composition of the BA pool was measured by GCMS and complemented with measurement of fecal BAs. Finally, gene expression of intestinal BA transporters (Fabp6, Slc10a2) were measured. Serum BAs were higher in -/- mice and composed of nor-BAs and bile alcohols which were absent in controls. Compared with controls, the BA pool of -/- mice was reduced 3-fold and enriched more than 30-fold (p<0.01) with nor-cholate whereas chenodeoxycholic acid was reduced 7-fold (p<0.01). Despite unchanged Abcc2 gene expression, bile flow was 1.5-fold higher (p<0.05) in -/- mice. BA output was reduced 68% (p<0.01) which correlated with a significant suppression of Abcb11 gene expression. Biliary excretion of cholesterol and phospholipids were unchanged in -/- mice and no significant change of Abcb4 gene expression could be detected. In contrast to controls, Slc10a1 gene expression was induced by 40% (p<0.05) whereas no significant changed was observed for Slc21a1. Fecal BA excretion was reduced to 50% (p<0.001) in -/- mice and bile alcohols were present -/- mice but not in controls. In all mice intestinal Slc10a2 and Fabp6 expression increased from proximal to distal. However, Slc10a2 and Fabp6 expression was significantly (p<0.01) lower in -/-mice. Our findings suggest that impaired peroxisomal BA synthesis is associated with adaptive changes of BA metabolism. More hydrophilic BAs of a smaller BA pool size are poor substrates for the BA export pump and explain the lower Abcb11 expression with a reduced BA output. This may further result in increased serum BA concentrations although we were unable to document inhibition of Slc10a1 or Slc21a1 expression. Decreased Slc10a2 and Fabp6 expression implies that conservation of the more hydrophilic BA pool was achieved by inducing BA absorption in more proximal parts of the intestine.

Targeted Deletion of the Ileal Bile Acid Transporter Eliminates Enterohepatic Cycling of Bile Acids in Mice

The ileal apical sodium bile acid cotransporter participates in the enterohepatic circulation of bile acids. In patients with primary bile acid malabsorption, mutations in the ileal bile acid transporter gene (Slc10a2) lead to congenital diarrhea, steatorrhea, and reduced plasma cholesterol levels. To elucidate the quantitative role of Slc10a2 in intestinal bile acid absorption, the Slc10a2 gene was disrupted by homologous recombination in mice. Animals heterozygous (Slc10a2+/-) and homozygous (Slc10a2-/-) for this mutation were physically indistinguishable from wild type mice. In the Slc10a2-/- mice, fecal bile acid excretion was elevated 10- to 20-fold and was not further increased by feeding a bile acid binding resin. Despite increased bile acid synthesis, the bile acid pool size was decreased by 80% and selectively enriched in cholic acid in the Slc10a2-/- mice. On a low fat diet, the Slc10a2-/- mice did not have steatorrhea. Fecal neutral sterol excretion was increased only 3-fold, and intestinal cholesterol absorption was reduced only 20%, indicating that the smaller cholic acid-enriched bile acid pool was sufficient to facilitate intestinal lipid absorption. Liver cholesteryl ester content was reduced by 50% in Slc10a2-/- mice, and unexpectedly plasma high density lipoprotein cholesterol levels were slightly elevated. These data indicate that Slc10a2 is essential for efficient intestinal absorption of bile acids and that alternative absorptive mechanisms are unable to compensate for loss of Slc10a2 function.

The SLC14 gene family of urea transporters.

Carrier-mediated urea transport allows rapid urea movement across the cell membrane, which is particularly important in the process of urinary concentration and for rapid urea equilibrium in non-renal tissues. Urea transporters mediate passive urea uptake that is inhibited by phloretin and urea analogues. Facilitated urea transporters are divided into two classes: (1) the renal tubular/testicular type of urea transporter, UT-A1 to -A5, encoded by alternative splicing of the SLC14A2 gene, and (2) the erythrocyte urea transporter UT-B1 encoded by the SLC14A1 gene. The primary structure of urea transporters is unique, consisting of two extended, hydrophobic, membrane-spanning domains and an extracellular glycosylated-connecting loop. UT-A1 is the result of a gene duplication of this two-halves-structure, and the duplicated portions are linked together by a large intracellular hydrophilic loop, carrying several putative protein kinase A (PKA) and -C (PKC) phosphorylation sites. UT-A1 is located in the apical membrane of the kidney inner medullary collecting duct cells, where it is stimulated acutely by cAMP-mediated phosphorylation in response to the antidiuretic hormone vasopressin. Vasopressin also up-regulates UT-A2 mRNA/protein expression in the descending thin limb of the loops of Henle. UT-A1 and UT-A2 are regulated independently and respond differently to changes in dietary protein content. UT-A3 and UT-A4 are located in the rat kidney medulla and UT-A5 in the mouse testis. The widely expressed UT-B participates in urea recycling in the descending vasa recta, as demonstrated by a relatively mild "urea-selective" urinary concentrating defect in transgenic UT-B null mice and individuals with the Jk(null) blood group.

Catalog of 238 variations among six human genes encoding solute carriers ( hSLCs) in the Japanese population.

We screened DNAs of 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in six genes encoding proteins of the solute carrier (SLC) family by direct sequencing of their entire genomic regions except for repetitive-sequence elements. This approach identified 213 SNPs and 25 insertion/deletion polymorphisms among the six genes. On average, we identified 1 SNP in every 509 nucleotides. Of the 213 SNPs, 14 were identified in the SLC10A1 gene, 51 in SLC15A1, 29 in SLC22A1, 27 in SLC22A2, 54 in SLC22A4, and 38 in SLC22A5. Eight were located in 5' flanking regions, 172 in introns, 25 in exons, and 8 in 3' flanking regions. These variants should contribute to investigations of possible correlations between genotypes and phenotypes as regards disease susceptibilities or responsiveness to drug therapy.

Characterization of the Genomic Structure of the Human Vitamin C Transporter SVCT1 (SLC23A2)

Vitamin C (L-ascorbic acid), a critical cofactor for intracellular enzymatic reactions, functions as a scavenger of free oxygen radicals and is an essential micronutrient. Vitamin C is actively transported into cells by one of two closely related sodium-dependent transporters, SVCT1 or SVCT2. In this paper, we report the complete sequencing and gene structure of SLC23A2, the gene encoding SVCT1. The1797-bp cDNA sequence (open reading frame) of the SLC23A2 gene was derived from a compact genomic sequence of 7966 bp [translation initiation codon (ATG) to poly A tail], which is divided into 14 exons. Furthermore, repetitive or masked elements constituted 17.98% of the gene; there were 4 Alu sequences and 5 MIR (Mammalian Interspersed Repetitive element) sequences. A search for common variants in SLC23A2, using current bioinformatic tools and direct resequencing of control populations, failed to identify common single nucleotide polymorphisms. The start of transcription was mapped to a position -47 relative to the ATG; the immediate 5' sequence was determined and analyzed for possible consensus binding sites for known transcription factors. Our findings will serve as the foundation for investigation of the regulation and expression of the tissue-specific sodium-dependent vitamin C transporter, SLC23A2.

Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene.

We have isolated and characterized the human homolog of the rat largest urea transporter of the UT-A family (hUT-A1). The 4.2-kb hUT-A1 cDNA encodes a 920-amino acid peptide, which is 89% identical to the rat UT-A1 protein. By Northern hybridization, hUT-A1 expression is detected in the human inner medulla as a approximately 4.4-kb mRNA transcript. By Western analysis, hUT-A1 is identified as a approximately 100-kDa protein in the human inner medulla. By immunohistochemistry, hUT-A1 expression is localized to the inner medullary collecting duct (IMCD). When transfected into HEK-293 cells hUT-A1 cDNA is translated into a approximately 98-kDa protein. Expression of hUT-A1 in Xenopus oocytes results in phloretin-inhibitable uptake of (14)C-urea, which shows only modest stimulation by cAMP, suggesting that in the human IMCD vasopressin may have a limited role in the short-term regulation of hUT-A1-mediated urea transport. We determined the organization of the human Slc14a2 gene and identified 20 exons distributed over approximately 67.5 kb on chromosome 18, from which hUT-A1 and the other human urea transporter, hUT-A2, are transcribed.