Cloning and expression of SLC10A4, a putative organic anion transport protein

INTRODUCTION Bile acids, the major solute in bile, are physiologically important for promoting bile flow and facilitating the absorption of dietary lipids[1,2]. Bile acids are also involved in cholesterol homeostasis, xenobiotic excretion, as well as apop…     

Mutation Screening and Functional Study of SLC26A4 in Chinese Patients with Congenital Hypothyroidism

Objective:Defects in the human solute carrier family 26 member 4 (SLC26A4) gene are reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify SLC26A4 mutations in Chinese patients with CH and analyze the function of the mutations.Methods:Patients with primary CH were screened for 21 CH candidate genes mutations by targeted next-generation sequencing. All the exons and exon-intron boundaries of SLC26A4 were identified and analyzed. The function of six missense mutation in SLC26A4 were further investigated in vitro.Results:Among 273 patients with CH, seven distinct SLC26A4 heterozygous mutations (p.S49R, p.I363L, p.R409H, p.T485M, p.D661E, p.H723R, c.919-2A>G) were identified in 10 patients (3.66%, 10/273). In vitro experiments showed that mutation p.I363L, p.R409H, p.H723R affect the membrane location and ion transport of SLC26A4, while p.S49R did not. Mutation p.T485M and p.D661E only affected ion transport, but had no effect on the membrane location.Conclusion:The prevalence of SLC26A4 mutations was 3.66% in Chinese patients with CH. Five mutations (p.I363L, p.R409H, p.T485M, p.D661E and p.H723R) impaired the membrane location or ion transport function of SLC26A4, suggesting important roles for Ile363, Arg409, Thr485, Asp661, and His723 residues in SLC26A4 function. As all variants identified were heterozygous, the pathogenesis of these patients cannot be explained, and the pathogenesis of these patients needs further study.     

Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. III: Role of solute and protein structure in proton-activated stilbenedisulfonate influx

DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) influx into magnesium resealed ghosts (MRSG) occurs over the anion/proton co-transport pH range (pK approximately 5.0). Here, factors are studied which may influence the pH dependence of DBDS transport. Accumulation of various stilbenedisulfonate (SD) molecules was studied and found to be correlated with the hydrophobicity of the R-groups (Hansch factor), not protonation of the sulfonates. The role of proton binding to glutamate 681 was found not to be part of the rate-limiting step in DBDS uptake by MRSG. Finally, the pH dependence of changes in quaternary structure/conformational state was investigated using an assay involving photo-crosslinking of band 3 subunits in the presence of DASD (4,4'-diazido-2,2'-stilbenedisulfonate). Lowering the pH promoted intersubunit crosslinking by DASD, with a pK value of 4.75+/-1.0. This value is comparable to the pK for DBDS binding to the "second" class of sites on control band 3 (pK = 5.01+/-0.16), and to DBDS influx into control MRSG (pK values between 4.57+/-0.15 and 4.7+/-0.1). Susceptibility to photo-crosslinking was reversed by raising the pH prior to initiation of the reaction. Significantly, no photo-crosslinking was observed between pH 6.0 and 8.0, where band 3 subunits are known to exist as stable dimers and tetramers. We conclude that intersubunit photo-crosslinking does not simply involve random collision between photo-activated DASD and band 3. Rather, proton binding to band 3 either alters the conformation at the interface between subunits of pre-existing tetramers, or it promotes self-association of stable dimers to a "novel" tetrameric conformational state.     

Effect of phenobarbital on the expression of bile salt and organic anion transporters of rat liver

BACKGROUND/AIMS: The hepatic clearance of drugs and cholephilic organic anions is stimulated by phenobarbital (PB). Our aim was to analyze the effects of PB on the expression of hepatocellular bile salt and organic anion transporters. METHODS: Male Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg/d) or saline for 5 days. Transporter expression was quantified by northern and western blot analysis and initial uptake rates of bromosulphophthalein (BSP) and digoxin were measured in isolated hepatocytes. RESULTS: Compared to control rats, PB treatment increased expression of the organic anion transporting polypeptide 2 (Oatp2; Slc21aS) more than 2-fold on the RNA (P < 0.05) and protein (P < 0.001) levels. Expression of Oatpl (Slc21al), Oatp4 (Slc21a6) and the Na+-taurocholate cotransporting polypeptide (Ntcp; Slc10a1) was unaltered. At the canalicular pole, expression of the bile salt export pump (Bsep; ABCB11) and of the multidrug resistance proteins 2 (Mrp2; ABCC2) and 6 (Mrp6; ABCC6) was not significantly changed. Whereas hepatocellular BSP uptake was unaffected by PB, digoxin uptake was stimulated 4-fold. CONCLUSIONS: The induction of digoxin uptake by PB correlates with Oatp2 expression. In contrast, the lack of increase of Oatpl and Oatp4 expression is in accordance with unchanged BSP uptake. These data challenge the previously held view that PB induces hepatocellular BSP uptake systems.     

Novel gain of function mutation in the SLC40A1 gene associated with hereditary haemochromatosis type 4

Here we report the case of a 69‐year‐old Chinese Han woman who presented with liver cirrhosis, diabetes mellitus, skin hyperpigmentation, hyperferritinaemia and high transferrin saturation. Subsequent genetic analyses identified a novel heterozygous mutation (p.Cys326Phe) in the SLC40A1 gene. This is the first report regarding a SLC40A1 mutation in the Chinese Han population and provides novel clinical evidence for the importance of p.Cys326 in SLC40A1 gene function.     

Characterization of a Functional Polymorphism in the 3' UTR of SLC6A4 and its Association With Drinking Intensity

Background:  The propensity for severe drinking is hypothesized to be regulated by differential expression of serotonin transporter gene (SLC6A4) in the human brain. The SLC6A4 promoter region 5-HTTLPR has been examined previously as a candidate polymorphic variant associated with severe drinking. In this study, we investigated whether other SLC6A4 single nucleotide polymorphisms (SNPs) are associated with drinking intensity among treatment-seeking alcoholics and whether these polymorphic variants result in differential SLC6A4 expression levels.
Methods:  We analyzed associations of drinking intensity in 275 (78.5% male) treatment-seeking alcoholics of Caucasian and Hispanic origin, with 6 SLC6A4 polymorphisms. Next, to examine the functionality of the SNP that showed a significant association with drinking intensity, we transfected the 2 alleles of rs1042173 into HeLa cell cultures and measured serotonin transporter mRNA and protein expression levels by using qRT-PCR and western blotting techniques.
Results:  One of the 6 polymorphisms we examined, rs1042173 in the 3' untranslated region (3'-UTR) of SLC6A4, showed a significant association with drinking intensity. The G allele carriers for rs1042173 were associated with significantly lower drinking intensity ( p  = 0.0034) compared to T-allele homozygotes. In HeLa cell cultures, the cells transfected with G allele showed a significantly higher mRNA and protein levels than the T allele-transfected cells.
Conclusion:  These findings suggest that the allelic variations of rs1042173 affect drinking intensity in alcoholics possibly by altering serotonin transporter expression levels. This provides additional support to the hypothesis that SLC6A4 polymorphisms play an important role in regulating propensity for severe drinking.     

Effects of statins on the adipocyte maturation and expression of glucose transporter 4 (SLC2A4): implications in glycaemic control

Aims/hypothesis Hyperlipidaemia often occurs in patients with type 2 diabetes mellitus. Though HMG-CoA reductase inhibitors (statins) are widely used for controlling hypercholesterolemia, atorvastatin has also been reported to have an adverse effect on glucose metabolism. Based on these findings, the aim of this study was to investigate the effects of statins on adipocytes, which play pivotal roles in glucose metabolism.Methods In 3T3-L1 cells, effects of statins on adipocyte maturation were determined morphologically. Protein and mRNA levels of SLC2A4 and adipocyte marker proteins were determined by immunoblotting and RT-PCR, respectively. Type 2 diabetic NSY mice were treated with atorvastatin for 15 weeks, followed by glucose and insulin tolerance tests and examination of SLC2A4 expression in white adipose tissue (WAT). Seventy-eight Japanese subjects with type 2 diabetes and hypercholesterolaemia were treated with atorvastatin (10 mg/day), and its effects on lipid and glycaemic profiles were measured 12 weeks after treatment initiation.Results Treatment with atorvastatin inhibited adipocyte maturation, SLC2A4 and C/EBPα expressions and insulin action in 3T3-L1 cells. Atorvastatin also attenuated SLC2A4 and C/EBPα expressions in differentiated 3T3-L1 adipocytes. These effects were reversed by l-mevalonate or geranylgeranyl pyrophosphate. In NSY mice, atorvastatin accelerated glucose intolerance as a result of insulin resistance and decreased SLC2A4 expression in WAT. In addition to improving hyperlipidaemia, atorvastatin treatment significantly increased HbA_1c but not fasting glucose levels in diabetic patients, and this effect was greater in the non-obese subgroup.Conclusions/interpretation These results demonstrate that atorvastatin attenuates adipocyte maturation and SLC2A4 expression by inhibiting isoprenoid biosynthesis, and impairs glucose tolerance. These actions of atorvastatin could potentially affect the control of type 2 diabetes.     

Impaired NFKBIE gene function decreases cellular uptake of methotrexate by down-regulating SLC19A1 expression in a human rheumatoid arthritis cell line

Objective: A non-synonymous single nucleotide polymorphism (nsSNP, rs2233434, Val194Ala) in the NFKBIE (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon) gene is known to be a rheumatoid arthritis (RA) susceptibility polymorphism in the Japanese RA population and could be closely associated with nuclear factor kappaB (NF-κB) activity. Inflammation caused by RA is sometimes associated with changes in expression levels of MTX (methotrexate) pathway-related genes. It is of interest to examine whether the NFKBIE gene had any influences on the mode of MTX action.

Methods: Both knockdown of NFKBIE gene expression and overexpression of wild-type NFKBIE and Val194Ala mutation were performed. A transfected human RA synovial cell line was cultured and then gene expressions in the MTX pathway were measured. In addition, we measured the uptake and efflux of MTX derivatives under the NFKBIE knockdown condition.

Results: Knockdown of NFKBIE reduced the mRNA for SLC19A1, a main MTX membrane transporter, and the intracellular accumulations of MTX derivatives. Moreover, our experiments also confirmed that overexpression of Val194Ala mutant NFKBIE decreased the SLC19A1 mRNA when compared to that of wild-type NFKBIE.

Conclusions: We suggest that the impairment of NFKBIE gene function can reduce the uptake of MTX into cells, suggesting that the gene is an important factor for the RA outcome.     

Risky alcohol use in adolescence: the role of genetics (DRD2, SLC6A4) and coping motives

Background: Drinking to cope (i.e., drinking to forget or alleviate negative feelings) has been found to be associated with adolescents’ heavy drinking and alcohol‐related problems. Additionally, it is widely accepted that genetic factors are involved in alcohol use and dependence. Studies are only beginning to reveal, however, which specific genotypes are related to drinking behaviors, and it is unknown whether they may interact with coping motives in predicting adolescents’ risky drinking. The aim of this study was to examine relationships between the dopamine D2 receptor gene (DRD2) Taq1A polymorphism (rs1800497), a serotonin transporter gene (SLC6A4) polymorphism (5‐HTTLPR), coping motives, and adolescents’ binge drinking and alcohol‐related problems. Methods: Participants in this cross‐sectional study were 282 Dutch adolescents (mean age 17.4, 47% men) who had consumed alcohol at least once in their life. Results: Coping motives were positively related to both binge drinking and alcohol‐related problems, while DRD2 and SLC6A4 genotypes were not. DRD2, but not the SLC6A4 genotype, interacted with coping motives. The link between coping motives and alcohol outcomes was stronger among those carrying the DRD2 risk (A1) allele. Conclusions: This study extends the present literature by providing additional insight into the etiological factors of adolescent drinking behavior. An interaction between a vulnerability gene (DRD2) and a cognitive factor (coping drinking) was found to be related to adolescents’ binge drinking and alcohol‐related problems.     

布鲁氏菌bp26和OMP10基因的原核表达和鉴定

目的研究布鲁氏菌外膜蛋白OMP10和bp26基因的表达、克隆与测序,并对其进行初步的血清学鉴定。方法从布鲁氏菌基因组中获得OMP10和bp26基因连接入PMD18-T克隆质粒并测序,克隆入融合表达载体PGEX-4T-1,构建重组质粒PGEX-4T-1/OMP10和PGEX-4T-1/bp26,在大肠杆菌中将该蛋白表达。用western-blot分析重组表达的GST-OMP10和GST-bp26的免疫学特性。结果成功构建了PGEX-4T-1/OMP10和PGEX-4T-1/bp26原核表达载体,并在大肠杆菌中成功表达了OMP10和bp26基因,布鲁氏菌免疫动物血清能特异性识别所表达的蛋白。结论布鲁氏菌外膜蛋白OMP10和bp26基因的成功表达,它可以与布鲁氏菌免疫血清产生特异性结合反应,为之后的抗原性以及免疫原性研究打下良好的物质基础。     

Membrane cation and anion transport activities in erythrocytes of hereditary spherocytosis: Effects of different membrane protein defects

Hereditary spherocytosis (HS) is due to different membrane protein defects (i.e., deficiency of spectrin and ankyrin, band 3, or band 4.2). In order to gain new insight into the relationships between band 3 function and proteins associated with the cytoskeleton, we studied erythrocyte anion transport activity in HS characterized by different membrane protein defects. Anion transport activity was increased in HS due to partial band 4.2 deficiency or to band 4.2 absence, while in HS associated with deficiency of spectrin + ankyrin or band 3, the anion transport results were normal or decreased, respectively. Moreover, since HS erythrocytes are characterized by an increased Na and a decreased K, we studied the principal membrane cation transport pathways. Activity of the Na/K pump was increased in all HS studied, while no changes in Na/K/2Cl cotransport and Na/Li exchange were evident between control and HS as well as between forms of HS associated with different membrane protein defects. K/Cl cotransport activity was decreased in all HS studied compared to normal red cells. In all HS, passive membrane permeability to Na and K was increased compared to normal erythrocytes. The increased Na and the low K content can be attributed to the abnormal membrane permeability to cations, which is not related to a specific membrane protein defect. Am. J. Hematol. 55:121–128, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat

BACKGROUND/AIMS: Alterations in hepatobiliary transporters may render fatty livers more vulnerable against various toxic insults. METHODS: We therefore studied expression and function of key organic anion transporters and their transactivators in 8-week-old obese Zucker rats, an established model for non-alcoholic fatty liver disease. RESULTS: Compared to their heterozygous littermates, obese animals showed a significant reduction in canalicular bile salt secretion, which was paralleled by significantly diminished Oatp2 mRNA and protein levels together with reduced nuclear HNF3beta, while expression of bile salt export pump, organic anion transporter (Oatp) 1 and multidrug resistance-associated protein (Mrp) 4 were unchanged. Impaired bile salt-independent bile flow in obese rats was associated with a 50% reduction of biliary secretion of the Mrp 2 model-substrates glutathione disulfide and S-(2,4-dinitrophenyl)glutathione. In line Mrp2 protein expression was reduced by 50% in obese rats. CONCLUSIONS: Oatp2 and Mrp2 expression is decreased in fatty liver and may impair metabolism and biliary secretion of numerous xenobiotics. Reduction of bile salt secretion and absence of biliary GSH excretion may contribute to impaired bile flow and posthepatic disorders associated with biliary GSH depletion.     

有机阴离子转运肽oatp4a1与脾主运化本质的关系

目的:通过观察脾虚状下大鼠oatp4a1蛋白表达来探讨oatp4a1与脾主运化本质的关系.方法:36只♂大鼠随机分为空白组、空白AA(AA为马兜铃酸Ⅰ)组、脾虚组、脾虚AA组、高脂饮食组、高脂饮食AA组6组.高脂饮食及利血平造模21 d,造模成功后给予相应组别大鼠AA灌胃3d,末次给AA后1h内采集标本,免疫组化方法检...     

人肝脏胆汁酸转运蛋白基因的克隆表达及其亚细胞定位

胆汁酸在脂肪和维生素消化吸收中发挥重要作用,当被排入小肠后约95%通过顶端钠依赖胆汁酸转运蛋白(ABST)被重吸收,其中的80%可通过牛磺胆汁酸钠共转运多肽(NTCP)被摄入肝细胞内并再次被分泌到胆汁中形成胆汁酸的肠肝循环[1].