ABCMdb

ABCB11 p.Arg432Thr

ClinVar:	c.1295G>C , p.Arg432Thr D , Pathogenic
Reviews:	p.Arg432Thr D
Predicted by SNAP2:	A: D (59%), C: D (53%), D: D (75%), E: D (71%), F: D (66%), G: D (63%), H: N (53%), I: D (59%), K: N (72%), L: D (59%), M: N (53%), N: N (53%), P: D (66%), Q: N (53%), S: N (57%), T: N (53%), V: D (59%), W: D (71%), Y: D (63%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Genetic variability, haplotype structures, and eth... Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8. Lang T, Haberl M, Jung D, Drescher A, Schlagenhaufer R, Keil A, Mornhinweg E, Stieger B, Kullak-Ublick GA, Kerb R
Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11).
Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8., [PMID:16763017]

Abstract [show]
Biliary excretion of bile salts and other bile constituents from hepatocytes is mediated by the apical (canalicular) transporters P-glycoprotein 3 (MDR3, ABCB4) and the bile salt export pump (ABCB11). Mutations in ABCB4 and ABCB11 contribute to cholestatic diseases [e.g., progressive familial intrahepatic cholestasis 2 (PFIC2), PFIC3, and intrahepatic cholestasis of pregnancy], and our objective was to establish genetic variability and haplotype structures of ABCB4 and ABCB11 in healthy populations of different ethnic backgrounds. All coding exons, 5 of 6 noncoding exons, 50 to 300 base pairs of the flanking intronic regions, and 2.5 to 2.8 kilobase pairs of the promoter regions of ABCB4 and ABCB11 were sequenced in 159 and 196 DNA samples of Caucasian, African-American, Japanese, and Korean origin. In total, 76 and 86 polymorphisms were identified in ABCB4 and ABCB11, respectively; among them, 14 and 28 exonic polymorphisms, and 8 and 10 protein-altering variants, of which 4 were predicted to have functional consequences. Both genes showed substantial ethnic differences with respect to allele number, frequency of common and population-specific sites, and patterns of linkage disequilibrium. Population genetic analysis suggested some selective pressure against changes in the protein, supporting the important endogenous role of these transporters. Haplotype variability was greater in ABCB11 than in ABCB4. An ABCB11 promoter haplotype was associated with significant decrease of activity compared with wild type. Our results contribute to a better understanding of the molecular basis and of ethnic differences in drug response, and provide a valuable tool for future research on the heredity of cholestatic liver injury.

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286 A splicing mutation (ϩ3)AϾC (intron 4) combined with a frameshift mutation in exon 22 (p.K930X), resulting in a PFIC2 phenotype and two nonsynonymous variants in exon 9 (p. E297G) and in exon 12 (p.R432T), were encountered in the patient exhibiting a BRIC (benign recurrent intrahepatic cholestasis) phenotype. Login to comment

[hide] The bile salt export pump. Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19. Stieger B, Meier Y, Meier PJ
The bile salt export pump.
Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19., [PMID:17051391]

Abstract [show]
Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver disease.

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160 Their bile flow rate is slightly but not significantly lower in comparison to controls, but the total bile salt output into bile is massively reduced and their liver bile salt concen- S114R G238V V284L* C336S D482G R487H S593R E636G G982R G1004D R1153CD R1268Q E186G E297G R432T I498T I498T T923P A926P R1050C R1128H S194P G260D N519S A1228V V444A K461E M677V R698H PFIC2 BRIC2 acquired cholestasis SNP Fig. 2 Putative secondary structure of Bsep (NT-005403) generated with the TOPO program (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl). Login to comment

[hide] Prediction of drug-induced intrahepatic cholestasi... Expert Opin Drug Saf. 2007 Jan;6(1):71-86. Sakurai A, Kurata A, Onishi Y, Hirano H, Ishikawa T
Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump.
Expert Opin Drug Saf. 2007 Jan;6(1):71-86., [PMID:17181454]

Abstract [show]
Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

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120 H2N COOH S56L G238V G260D C336S L339V V444A K461E D482G T923P K930X G982R R1090X R1153C Outside Inside R1268Q A1228VE1186K R1128H R1057X R1050C A926P A865V R698H E636G M677V S593R E592Q N591S R575XA570T Q558H I498T R432T R415Q R299K E297G V284A I206V S194P E186G cholestasis Expert Opin. Drug Saf. (2007) 6(1) Table 1. Login to comment
121 Nonsynonymous polymorphisms and mutations in the ABCB11 gene NCBI No. Exon Nucleotide Amino acid alteration Phenotype Ref. Position Alteration rs11568361 5 167 C→T Ser56Leu - [102] - 5 341 G→C Ser114Arg PFIC2 [47]* - 6 557 A→G Glu186Gly BRIC2 [45,48] - 6 580 T→C Ser194Pro - [44] rs11568358 7 616 A→G Ile206Val - [102] - 7 695 T→del Frame shift at position 232 PFIC2 [47] - 7 713 G→T Gly238Val PFIC2 [47] - 8 779 G→A Gly260Asp - [44] - 8 851 T→C Val284Ala - [44] rs11568372 8 890 A→G Glu297Gly PFIC2/BRIC2 [35,43,45,47,102] rs2287617 8 896 G→A Arg299Lys - [102] - 8 908 G→del Frame shift at position 303 PFIC2 [35] - 9 1007 G→C Cys336Ser PFIC2 [47] - 9 1015 C→G Leu339Val - [46] - 11 1244 G→A Arg415Gln - [39] - 11 1294 G→C Arg432Thr BRIC2 [43] rs2287622 12 1331 T→C Val444Ala ICP/PFIC2? Login to comment

[hide] Missense mutations and single nucleotide polymorph... Hepatology. 2009 Feb;49(2):553-67. Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, Thompson RJ
Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing.
Hepatology. 2009 Feb;49(2):553-67., [PMID:19101985]

Abstract [show]
The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single-nucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency.

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67 ABCB11 Missense Mutations and SNPs Functionally Analyzed in This Study Exon Nucleotide Change Predicted Protein Effect Location in Protein Associated Phenotype Prevalence or Frequency* Any Defect(s) Identified Reference 4 c.149TϾC L50S NH2 term PFIC 1 family (het) Immature protein 31 5 c.270TϾC F90F EC1 SNP 2.7%-7.7% 43, 45 6 c.403GϾA E135K EC1 BRIC 1 family (het) Reduced levels of mature protein † 6 c.409GϾA E137K EC1 BRIC / ICP 1 family (het) Immature protein ‡ 7 c.500CϾT A167V TM2 PFIC 1 family (hom) Mild exon skipping beta 7 c.557AϾG E186G IC1 BRIC 2 families (both het) Moderate exon skipping; greatly reduced levels of mature protein 8, 37 7 c.580TϾC S194P IC1 SNP-PSC 1.1% 43 7 c.593TϾC L198P IC1 BRIC / ICP / DC 1 family (het) Greatly reduced levels of mature protein # 8 c.713GϾT G238V EC2 PFIC 1 family (hom) 29 8 c.725CϾT T242I TM4 PFIC 1 family (het) 31 8 c.779GϾA G260D TM4 SNP-PBC 0.8% 43 9 c.850GϾC V284L IC2 PFIC 1 family (het) No protein 28 9 c.851TϾC V284A IC2 SNP 0.5% Increased levels of mature protein 43, 45† 9 c.889GϾA E297K IC2 Prolonged NNH 1 family (het) Moderate differential splicing; immature protein ‡ 9 c. 890AϾG E297G IC2 PFIC, BRIC PFIC, 45 families (14 hom, 31 het) BRIC, 4 families (2 hom, 2 het) Greatly reduced levels of mature protein 7, 8, 12, 29-32, 35 10 c.936GϾT Q312H IC2 PFIC 1 family (het) ‡ 10 c.937CϾA R313S IC2 PFIC 1 family (het) 31 10 c.957AϾG G319G TM5 SNP 1.5 - 7.5% Mild exon skipping 42, 43, 45 10 c.980GϾA G327E TM5 PFIC 1 family (het) 31 10 c.1007GϾC C336S TM5 PFIC 1 family (het) 29 11 c.1168GϾC A390P NBF PFIC, BRIC 2 families (both het) Immature protein 31; # 12 c.1129GϾA G410D NBF PFIC 1 family (het) 31 12 c.1238TϾG L413W NBF PFIC 1 family (het) Greatly reduced levels of mature protein 31 12 c.1244GϾA R415Q NBF SNP-ICP 1.3% 42 12 c.1295GϾC R432T NBF BRIC 1 family (het) Reduced levels of mature protein 12 13 c.1331CϾT A444V NBF SNP, ICP, CC, DC, BRIC 43-60% Increased levels of mature protein 8, 28, 37, 39-45 13 c.1381AϾG K461E WA PFIC 1 family (hom) Immature protein 7 13 c.1388CϾT T463I WA PFIC 1 family (het) Mild exon skipping 31 13 c.1396CϾA Q466K Adj WA PFIC 1 family (het) 31 13 c.1409GϾA R470Q Adj WA PFIC 2 families (1 het, 1 consanguineous) Immature protein 31 14 c.1442TϾA V481E NBF1 PFIC 1 family (het) 31 14 c.1445AϾG D482G NBF1 PFIC 22 families (16 het, 6 hom) Severe differential splicing; immature protein 7, 30-32 14 c.1468AϾG N490D NBF1 PFIC 1 family (het) Greatly reduced levels of mature protein; reduction in bile salt transport 31 14 c.1493TϾC I498T NBF1 PFIC / BRIC 1 family (het) 38 14 c.1530CϾA T510T NBF1 SNP-PBC 0.7% 43 14 c.1535TϾC I512T NBF1 PFIC 1 family (het) 31 14 c.1544AϾC N515T NBF1 PFIC 1 family (het) 31, 32 14 c.1440GϾA R517H NBF1 PFIC 1 family (het) No protein 31, 32 14 c.1605CϾT A535A NBF1 SNP 0.3% Slightly reduced levels mature protein 39, 45 14 c.1621AϾC I541L NBF1 PFIC 3 families (1 het, 2 consanguineous) No protein 31-33 15 c.1643TϾA F548Y Adj ABCm PFIC 1 family (het) 31, 32 15 c.1685GϾA G562D ABCm PFIC 1 family (het) 31 15 c.1708GϾA A570T Adj ABCm/WB PFIC, BRIC PFIC, 1 family Greatly reduced levels of mature protein; reduction in bile salt transport 8, 31 Table 1. Login to comment
141 Mature BSEP, but at somewhat reduced levels, was detected for E135K (c.403GϾA; Fig. 5B), T655I (c.1964CϾT; Fig. 5C), R432T (c.1295GϾC; Fig. 5.e), the PFIC-associated SNP Y818F (c.2453AϾT; Fig. 5E), and the SNP A535A (c.1605CϾT; Fig. 5F). Login to comment

[hide] Role of the bile salt export pump, BSEP, in acquir... Drug Metab Rev. 2010 Aug;42(3):437-45. Stieger B
Role of the bile salt export pump, BSEP, in acquired forms of cholestasis.
Drug Metab Rev. 2010 Aug;42(3):437-45., [PMID:20028269]

Abstract [show]
Generation of bile is a key function of the liver. Its impairment leads to accumulation of cytotoxic bile salts in hepatocytes and, consequently, to liver disease. The bile salt export pump, BSEP, is critically involved in the secretion of bile salts into bile. Its function can be disturbed or abolished by inherited mutations. This will lead to progressive intrahepatic cholestais and severe liver disease. In addition to mutations, BSEP can be inhibited by acquired factors, such as xenobiotics or drugs, aberrant bile salt metabolites, or pregnancy. This inhibition will lead to acquired cholestasis. Some drugs are now known to be competitive inhibitors of Bsep. In addition, a polymorphism in the gene coding for BSEP has been identified as a potential susceptibility factor for acquired cholestasis.

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78 For example, in a patient with benign recurrent intrahepatic cholestasis type 2, the two compound heterozygous mutations, E297G and R432T, were identified. Login to comment

[hide] Xenobiotic, bile acid, and cholesterol transporter... Pharmacol Rev. 2010 Mar;62(1):1-96. Epub 2010 Jan 26. Klaassen CD, Aleksunes LM
Xenobiotic, bile acid, and cholesterol transporters: function and regulation.
Pharmacol Rev. 2010 Mar;62(1):1-96. Epub 2010 Jan 26., [PMID:20103563]

Abstract [show]
Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.

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6508 Nucleotide Change Amino Acid Change In Vitro Function Protein Expression/ Localization ABCB11 BSEP N.D. G238V N.D. Intracellular A890G E297G 2 Intracellular N.D. C336S ↔ Normal G1296C R432T 2 Reduced T1331C V444A ↔ Normal/Reduced A1445G D482G 2 Normal/Reduced G2026T D676Y 2 Reduced G2563A G855R 2 Reduced G2944A G982R 2 Intracellular C3457T R1153C 2 Intracellular G3803A R1268Q 2 Intracellular searchers were able to identify functional roles for Mrp2 using rats lacking this transporter (Eisai hyperbilirubinemic rats on a Sprague-Dawley background and transport-deficient (TR-) on a Wistar background) (Paulusma et al., 1996; Ito et al., 1997). Login to comment

[hide] Combined mutations of canalicular transporter prot... Gastroenterology. 2006 Aug;131(2):624-9. Keitel V, Vogt C, Haussinger D, Kubitz R
Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy.
Gastroenterology. 2006 Aug;131(2):624-9., [PMID:16890614]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder that usually develops in the third trimester of pregnancy and persists until delivery. The cause of ICP remains elusive, but there is evidence that mutations in the canalicular ABC transporter phospholipid flippase (MDR3) and in the bile salt export pump (BSEP) can predispose for the development of ICP. MDR3 and BSEP were investigated by gene sequencing and immunofluorescence microscopy in a patient with severe ICP of early onset. ICP was diagnosed in a patient in the first trimester of pregnancy with severe pruritus, elevated levels of bile salts, and 48-fold elevation of transaminase levels. A liver biopsy specimen showed diminished canalicular expression of the bile salt export pump BSEP, while the expression and localization of the phospholipid flippase MDR3 was normal. Gene sequencing revealed a homozygous MDR3 gene mutation (S320F). The patient was also homozygous for the common BSEP polymorphism V444A. Treatment with ursodeoxycholate normalized transaminase levels but could not prevent further elevation of bile salt levels and preterm delivery. The combined homozygous alterations of the canalicular transporters may explain the early onset and severity of ICP in this patient. The common BSEP polymorphism V444A accounts for the reduced canalicular BSEP expression. Reduced bile salt secretion through BSEP may explain the persistence of elevated bile salt levels and incomplete efficacy of ursodeoxycholate treatment.

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79 In addition to changes in protein localization, V444A may alter transport activity as described recently for 2 BRIC-associated BSEP mutations (R432T and E297G).19 Another possibility of reduced transporter activity includes an increased susceptibility of V444A toward the inhibitory effect of estrogens.20 Homozygous V444A can be expected in 25% of the population, but homozygous V444A alone cannot explain the severity of ICP in our patient. Login to comment

[hide] Enterohepatic bile salt transporters in normal phy... Gastroenterology. 2004 Jan;126(1):322-42. Kullak-Ublick GA, Stieger B, Meier PJ
Enterohepatic bile salt transporters in normal physiology and liver disease.
Gastroenterology. 2004 Jan;126(1):322-42., [PMID:14699511]

Abstract [show]
The vectorial transport of bile salts from blood into bile is essential for the generation of bile flow, solubilization of cholesterol in bile, and emulsification of lipids in the intestine. Major transport proteins involved in the enterohepatic circulation of bile salts include the hepatocellular bile salt export pump (BSEP, ABCB11), the apical sodium-dependent bile salt transporter (ASBT, SLC10A2) in cholangiocytes and enterocytes, the sodium-dependent hepatocyte bile salt uptake system NTCP (SLC10A1), the organic anion transporting polypeptides OATP-C (SLC21A6), OATP8 (SLC21A8) and OATP-A (SLC21A3), and the multidrug resistance protein MRP3 (ABCC3). Synthesis and transport of bile salts are intricately linked processes that undergo extensive feedback and feed-forward regulation by transcriptional and posttranscriptional mechanisms. A key regulator of hepatocellular bile salt homeostasis is the bile acid receptor/farnesoid X receptor FXR, which activates transcription of the BSEP and OATP8 genes and of the small heterodimer partner 1 (SHP). SHP is a transcriptional repressor that mediates bile acid-induced repression of the bile salt uptake systems rat Ntcp and human OATP-C. A nuclear receptor that activates rodent Oatp2 (Slc21a5) and human MRP2 (ABCC2) is the pregnane X receptor/steroid X receptor PXR/SXR. Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases in response to physiologic stimuli such as cyclic adenosine monophosphate or taurocholate. Finally, dysfunction of individual bile salt transporters such as BSEP, on account of genetic mutations, steric inhibition, suppression of gene expression, or disturbed signaling, is an important cause of cholestatic liver disease.

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119 A clinical syndrome with recurrent intrahepatic cholestasis but normal liver architecture in an adolescent patient has been associated with compound heterozygosity for the E297G and a novel R432T mutation,176 suggesting that certain adult forms of cholestasis may also be caused by BSEP mutations and reduced transport function. Login to comment
141 Role of Bile Salt Transporters in the Pathogenesis of Liver Disease Species Transport protein Gene symbol Physiologic function Alterations in liver disease References Basolateral transport proteins Rat Ntcp Slc10a1 Naϩ-dependent hepatocellular bile salt uptake Decreased expression in rat models of cholestasis Decreased mRNA and protein levels during pregnancy, associated with decreased nuclear binding of HNF1␣ and RAR␣:RXR␣ 201,211,231 232 Human NTCP SLC10A1 Naϩ-dependent hepatocellular bile salt uptake Decreased mRNA and protein levels in human cholestatic liver disease 30,187 Decreased expression in HCC 72 Rat Oatp1 Slc21a1 Multispecific uptake of organic anions and amphipathic compounds Decreased expression in bile duct ligation and in ethinyl estradiol induced cholestasis 211,233 Oatp2 Slc21a5 Multispecific uptake of organic anions and of cardiac glycosides (digoxin) Decreased mRNA but not protein levels in carbon tetrachloride induced liver injury Decreased mRNA and protein levels in ethinylestradiol-induced cholestasis 234 126 Oatp4 Slc21a10 Multispecific uptake of organic anions and amphipathic compounds Decreased expression in bile duct ligation and sepsis 235 Human OATP-C SLC21A6 Hepatocellular uptake of bile salts and other organic anions Reduced mRNA in PSC and inflammatory cholestasis Decreased expression in HCC 29,30 217 OATP8 SLC21A8 Hepatocellular uptake of organic anions, peptides, and xenobiotics Decreased expression in HCC because of increased expression of the transcriptional repressor HNF3beta 218 Rat/human Mrp1/MRP1 ABCC1 Efflux of cytotoxic cations and non-bile salt organic anions Increased expression in hepatoma cells and sepsis 199,236 Rat/human Mrp3/MRP3 ABCC3 Efflux of organic anions, bile salts, and anticancer agents Increased expression in Eisai Hyperbilirubinemic Rats and in bile duct ligation Increased expression in Dubin-Johnson syndrome and primary biliary cirrhosis 237 61 Hepatocyte canalicular transport proteins Mouse/rat/ human Bsep/Bsep/ BSEP ABCB11 Canalicular efflux of bile salts Gene mutations and absence of the protein in patients with PFIC2, characterized by low GGT levels and reduced biliary bile acid excretion 174,238 Compound heterozygosity for the E297G/R432T mutations in a patient with recurrent intrahepatic cholestasis 176 Reduced mRNA and canalicular BSEP staining in human inflammatory cholestasis 30 Cisinhibition by cholestatic drugs such as cyclosporine A 190 Transinhibition by the cholestatic estrogen metabolite estradiol-17beta-D-glucuronide 190,239 Increased expression in C57L/J gallstone-susceptible mice, despite reduced bile salt excretory capacity 220,240 Mouse/rat/ human Mdr2/Mdr2/ MDR3 ABCB4 Biliary excretion of phospholipids Mdr2 -/- knockout mice exhibit an absence of phospholipids in bile and develop progressive liver disease with portal inflammation, bile duct proliferation and fibrosis 241 PFIC3, characterized by high GGT levels and absent lipoprotein X in serum, is caused by mutations in the MDR3 gene (chromosome 7q21) 177 MDR3 mutations in PFIC3 are associated with intrahepatic cholestasis of pregnancy 242 Rat/human Mrp2/MRP2 ABCC2 Canalicular excretion of organic anions Decreased mRNA and protein levels in bile duct ligation and endotoxinemia 200,243 Decreased canalicular density of Mrp2 transporter molecules in endotoxinemia, taurolithocholate cholestasis, and bile duct ligation 145,200,243 Mutations in the rat Mrp2 gene cause hereditary conjugated hyperbilirubinemia 244 Mutations in the human MRP2 gene cause the Dubin-Johnson syndrome with absent protein expression 181,183 MRP2 function is inhibited by anabolic 17␣-alkylated steroids 245,246 Decreased canalicular MRP2 staining in PBC and inflammatory cholestasis 30,31 Decreased mRNA levels in PSC 29 Human FIC1 ATP8B1 Putative aminophospholipid translocator P-type ATPase, positional candidate in genetic linkage analysis of PFIC1 (Byler`s disease) and BRIC 171 PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis. Login to comment

[hide] Functional analysis of nonsynonymous single nucleo... Pharmacogenet Genomics. 2008 Sep;18(9):823-33. Kobayashi K, Ito K, Takada T, Sugiyama Y, Suzuki H
Functional analysis of nonsynonymous single nucleotide polymorphism type ATP-binding cassette transmembrane transporter subfamily C member 3.
Pharmacogenet Genomics. 2008 Sep;18(9):823-33., [PMID:18698235]

Abstract [show]
OBJECTIVES: The multidrug resistance-associated protein 3/ATP-binding cassette transmembrane transporter subfamily C member 3 (MRP3/ABCC3) plays an important role in exporting endogenous and xenobiotic anionic substrates, including glucuronide conjugates of xenobiotics, from hepatocytes into the blood circulation. This excretory function of ABCC3 becomes very apparent particularly under cholestatic conditions, since ABCC3 is induced when the biliary excretion pathway is impaired. In this study, we analyzed the functional properties of 11 nonsynonymous single nucleotide polymorphisms (SNPs) in the ABCC3 gene found in the public SNP database. METHODS: HeLa and Sf9 insect cells were used to analyze the protein expression and transport function, respectively. RESULTS: After transient transfection of cDNA into HeLa cells, it was found that R1381S ABCC3 exhibits intracellular accumulation of immature protein, the localization of which was mostly merged with a marker for the endoplasmic reticulum. Two kinds of SNPs type ABCC3 (S346F and S607N) lost their transport activity for [H]estradiol-17beta-D-glucuronide in membrane vesicles from Sf9 cells infected with the recombinant baculoviruses, although the band length and the amount of protein expression remained normal. In contrast, the cellular localization, protein expression and function of other eight kinds of SNPs type ABCC3 (G11D, R99Q, V765L, P920S, R923Q, R1286G, R1348C, and Q1365R ABCC3) remained normal. CONCLUSION: The results of this study suggest that the possession of R1381S, S346F, and S607N types of ABCC3 sequences may be a possible risk factor for the acquisition of hepatotoxicity, due to their poor ability to transport toxic compounds across the sinusoidal membrane.

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173 For example, E297G ABCB11 is mislocalized to ER when heterologously expressed in MDCK cells [39,40], although the patient with compound heterozygosity for E297G and R432T ABCB11 showed normal canalicular localization, and the primary cause of dysfunction of ABCB11 was attributed to the reduced transport function [41]. Login to comment

[hide] Impaired expression and function of the bile salt ... J Hepatol. 2005 Sep;43(3):536-43. Noe J, Kullak-Ublick GA, Jochum W, Stieger B, Kerb R, Haberl M, Mullhaupt B, Meier PJ, Pauli-Magnus C
Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis.
J Hepatol. 2005 Sep;43(3):536-43., [PMID:16039748]

Abstract [show]
BACKGROUND/AIMS: Inherited dysfunction of the bile salt export pump BSEP (ABCB11) causes a progressive and a benign form of familial intrahepatic cholestasis, denominated as PFIC2 and BRIC2, respectively. We functionally characterized novel ABCB11 mutations encountered in two patients with a PFIC2 and a BRIC2 phenotype, respectively. METHODS: BSEP expression was determined in liver biopsies by immunohistochemistry. ABCB11 mutations were functionally characterized by taurocholate transport in SF9 cells transfected with human ABCB11. RESULTS: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((+3)A > C) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Hepatic BSEP expression was absent in PFIC2 and preserved in BRIC2. In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. CONCLUSIONS: The intron 4 (+3)A > C, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. By combining functional in-vitro characterization with immunohistochemical detection of variant BSEP we provide direct evidence for the role of ABCB11 mutations in the pathogenesis of different forms of intrahepatic cholestasis.

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4 Results: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((C3)AOC) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Login to comment
6 In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. Login to comment
7 Conclusions: The intron 4 (C3)AOC, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. Login to comment
61 The single nucleotide polymorphisms 890AOG (codon GAGOGGG) and 1294GOC (codon AGAOACA), which result in the nonsynonymous mutations E297G and R432T, respectively, were introduced into the reference h ABCB11 cDNA. Login to comment
68 Functional transport studies ATP-dependent transport of labeled bile salts was determined for the E297G and the R432T mutations by a rapid filtration assay as described [16]. Login to comment
100 The parents of the patient had normal liver enzyme levels in the absence of clinical signs of hepatopathy. 3.2.1. Sequencing Sequence analysis indicated a nonsynonymous mutation in exon 9 (891GOA) predicting the substitution of a glutamate by a glycine in position 297 (E297G) combined with a nonsynonymous mutation in exon 12 (1296GOC), predicting a substitution of an arginine by a threonine in position 432 (R432T). Login to comment
101 Analysis of the patient`s parents indicated that the E297G mutation was inherited from the mother, whereas the R432T mutation was inherited from the father (Fig. 1). Login to comment
110 Transport studies Western blot analysis detected comparable protein amounts for R432T, E297G and reference BSEP in SF-9 cell vesicles (Fig. 3). Login to comment
111 Transport capacity of the mutated constructs amounted to about 13 and 20% of reference activity for the R432T and the E297G construct, respectively (Fig. 3). Login to comment
112 Initial taurocholate transport by reference BSEP as well as by R432T and E297G mutants exhibited saturability with increasing substrate concentrations (Fig. 4). Login to comment
114 The Km values for R432T and the E297G mutant were 5.6 mmol/L and 22 mmol/L, respectively, which is comparable to the Km value of reference BSEP. Login to comment
115 In contrast, the maximum taurocholate transport velocity for the BSEP mutants was greatly reduced, with Vmax values of 53 pmol/L mg proteinK1 minK1 , and 111 pmol/ L mg proteinK1 minK1 for R432T and E297G, respectively, compared to 686 pmol/L mg proteinK1 minK1 for reference BSEP (Fig. 4). Login to comment
127 Compound heterozygosity for two nonsynonymous variants in exon 9 (E297G) and in exon 12 (R432T) was encountered in the patient exhibiting a BRIC phenotype. Login to comment
128 While the E297G site had already been associated with inherited intrahepatic cholestasis, the R432T mutation was previously undescribed in cholestatic disease. Login to comment
133 However, it cannot completely be excluded that in our patient the R432T allele is compensating for defective BSEP expression associated with the E297G mutation. Login to comment
146 Kinetics of ATP-dependent transport of taurocholate by reference BSEP, R432T BSEP mutant and E297G BSEP mutant in membrane vesicles isolated from Sf9 cells. Login to comment
147 Initial uptake rates for taurocholate by reference BSEP, E297G mutant (60 s) and R432T mutant (90 s) were determined in the presence and absence of ATP (5 mmol/L). Login to comment

[hide] The bile salt export pump (BSEP) in health and dis... Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12. Kubitz R, Droge C, Stindt J, Weissenberger K, Haussinger D
The bile salt export pump (BSEP) in health and disease.
Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12., [PMID:22795478]

Abstract [show]
The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.

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182 Mutations such as p.E186G, p.E297G, p.R432T, p.A570T, p.T923P, p.A926P, p.G1004D, p.R1050C and p.R1128H have been described in BRIC-2 patients [137,140-142] (Table 1). Login to comment
185 PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Missense mutations M1V C336S D549V L1055P E135K E137K T87R V43I S701P G19R W342G G556R C1083Y E137K L198P M123T S56L L712L L50S A382G G562D A1110E E186G E297G S194P Q121K A865D M62K R387H A570T S1114R L198P R415Q L198P R128H A865G C68Y A390P L581F G1116E E297G V444A G260D I206V S874P C107R G410D A588V G1116F G374S D482G E297K V284A I939M I112T L413W S593R G1116R A390P N591S V444A G295C R958Q W114R I420T I627T S1120N R432T T655I T510T G295R F959C Y157C D440E E636G R1128C V444A T655I G295S F959V A167T G455E R698C S1144R I498T D676Y R299K T965S A167V K461E S699P R1153C A570T P710P R303K F971L I182K T463I E709K R1153H T586I L827I L339V F971Y M183T Q466K G758R S1154P G648V G855R H423R L1006F M183V R470Q G766R N1173D T655I E1186K V444A N1009H G188W Y472C Y818F T1210P T923P V444D K1145N M217R V481E R832C N1211D A926P V444G I1183T R223C D482G R832H V1212F R948C A459V S226L R487H T859R R1231Q G1004D I468I G238V R487P A865V R1231W R1050C R487L T242I N490D Q869P L1242I G1116R Q546K A257G I498T G877R D1243G R1128H Q558H V284L G499E S901R R1268Q L1197G E592Q E297G I512T R948C A1283V R1231Q V597M R303G N515T N979D G1292V R616G R303K R517H G982R G1298R T619A Q312H F540L G1004D M677L R313S I541L T1029K M677V G327E I541T G1032R R696Q W330R F548Y A1044P R698H Nonsense mutations (premature stop-codons) S25X Y472X Y772X R1090X E96X W493X Q791X V1147X W330X R520X R928X Q1215X Y354X I528X Y1041X R1235X R415X R575X R1057X E1302X R470X Q702X Q1058X Table 1 (Continued) PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Splice site mutations 76 + 3G > T 908 + 1delG 2178 + 1G > T 3057-2A > G Q159Q 77-1G > C 908 + 1G > T 2179-2A > G 3213 + 1delG Q361Q 99-1G > T 908 + 1G > A 2343 + 1G > T 3213 + 4A > G 150 + 3A > C 1435-13 -8del 2343 + 2T > C 3213 + 5G > A 390-1G > A 2012-8T > G 2611-2A > T 611 + 1G > A 2178 + 1G > A R1001R Deletions/insertions/frame shifts Q101Dfs8X L380Wfs18X G648Vfs5X Q1058Hfs38X F959Hfs1X T127Hfs6X A382 A388del K700Sfs12X I1061Vfs34X F959Gfs48X N199Ifs14X P456Pfs24X T919del L1165del L232Cfs9X H484Rfs5X K930Efs92X A1192Efs50X R303Sfs17X I528Sfs21X K930Efs79X T1256Tfs40X V368Rfs27X I610Qfs45X K969 K972del Synonymous variants without disease association R33R F90F L232L I416I G557G I876I A1028A K1145K D36D I134I Y269Y G418G V597V G937G K1070K R52R S136S Q312Q F427F A804A Y981Y T1086T D58D V195V G319G E395E A535A G817G G1004G A1110A The overview shows ࣈ 290 known variants of BSEP on the protein level, except splice site mutations, which are shown on cDNA level. Login to comment

[hide] Differential effects of membrane cholesterol conte... Mol Pharmacol. 2014 Jun;85(6):909-20. doi: 10.1124/mol.114.092262. Epub 2014 Apr 7. Guyot C, Hofstetter L, Stieger B
Differential effects of membrane cholesterol content on the transport activity of multidrug resistance-associated protein 2 (ABCC2) and of the bile salt export pump (ABCB11).
Mol Pharmacol. 2014 Jun;85(6):909-20. doi: 10.1124/mol.114.092262. Epub 2014 Apr 7., [PMID:24711118]

Abstract [show]
Rat canalicular membranes contain microdomains enriched in cholesterol and ATP-binding cassette transporters. Cholesterol is known to regulate the activity of transporters. Here, we investigated the effect of membrane cholesterol on the transport kinetics of multidrug resistance-associated protein 2 (MRP2) and of bile salt export pump (BSEP) variants and mutants. MRP2 and BSEP were expressed with baculoviruses in insect cells, followed by vesicle isolation from control and cholesterol-loaded cells (1 mM cholesterol@randomly methylated-beta-cyclodextrin) for transport assays. We found that cholesterol stimulates MRP2 transport activity for substrates of different molecular weights: estradiol-17-beta-glucuronide (E17betaG), prostaglandin E2 (PGE2), cholecystokinin 8 (CCK8), and vasopressin displayed an increase of Vmax and a variable decrease of Km. Kinetics of E17betaG showed a sigmoidal shape and a mild cooperativity in Hanes-Woolf plots in control membranes. High cholesterol content shifted E17betaG to Michaelis-Menten kinetics. PGE2/glutathione transport followed Michaelis-Menten kinetics irrespective of cholesterol. The MRP2 substrates CCK8 and vasopressin exhibited Michaelis-Menten kinetics independent of membrane cholesterol content. Transport of ochratoxin A was ATP-dependent but was neither mediated by MRP2 nor stimulated by cholesterol. Transport of the two most common BSEP variants p.444V/A showed Michaelis-Menten kinetics irrespective of membrane cholesterol, whereby cholesterol leads to an increased Vmax while Km remains unchanged. The transport activity of the BSEP mutants p.E297G and p.R432T increased at high cholesterol content but did not reach the capacity of normal BSEP. Hence, changing membrane cholesterol content modulates BSEP and MRP2 transport kinetics differently. Cholesterol increases the transport rates of BSEP and MRP2, but with the latter, may also modify the binding site as for E17betaG.

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11 The transport activity of the BSEP mutants p.E297G and p.R432T increased at high cholesterol content but did not reach the capacity of normal BSEP. Login to comment
177 Finally, we studied the effect of cholesterol on two mutant forms of BSEP, p.E297G and p.R432T, found in patients with BSEP deficiency syndromes. Login to comment
262 (A, D, and G) Western blot analysis of p.444A variant (A), p.E297G (D), and p.R432T (G) mutant BSEP-expressing vesicles with and without cholesterol loading (30 mg protein per lane). Login to comment

[hide] Genetic variations of bile salt transporters. Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006. Kubitz R, Droge C, Kluge S, Stindt J, Haussinger D
Genetic variations of bile salt transporters.
Drug Discov Today Technol. 2014 Jun;12:e55-67. doi: 10.1016/j.ddtec.2014.03.006., [PMID:25027376]

Abstract [show]
Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTalpha/beta and discusses approaches for transporter analysis.

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137 BSEP/Bsep NTCP ASBT Exon skipping E186G G1116R G319G R1128C T463I R1128H A926P E1186K A1028Aa R1231W A1110E Aberrant splicing E297K R1153H R832C S1154P S1144R No splice product T586I R1231Q Reduced plasma membrane expression E135K A570T I223T E297Gb N591Sb V444A R1050C Intracellular retention Y818F G982R Reduced or absent bile salt transport A570T R432T A64T K314E V98Ic M264V I206V Q558H I223T C144Y P290S E297Gb N591Sb S267F L243P G374S E1186K I279T T262M a A1028A induces significant exon skipping in vitro but probably not in vivo (unpublished data; Dro &#a8;ge, Ha &#a8;ussinger, Kubitz). Login to comment