Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.

Abstract

Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…The bile acid transporters NTCP (SLC10A1) and ASBT (SLC10A2) NTCP As the first member of the SLC10 family, the Na + / taurocholate cotransporting polypeptide (Ntcp; Slc10a1) was isolated from rat liver mRNA by expression cloning using the Xenopus laevis oocytes expression system (Hagenbuch et al 1990(Hagenbuch et al , 1991. Further orthologs were subsequently cloned from human , mouse (Cattori et al 1999) and rabbit livers.…”

“…Membrane insertion and sorting of NTCP and ASBT Based on bioinformatic predictions and experimental data, it has been shown that NTCP and ASBT have an extracellular N-terminus, an odd number of transmembrane helices (seven or nine) and a cytoplasmic Cterminus (Hagenbuch et al 1991;Hagenbuch and Meier 1994;Stieger et al 1994;Dawson and Oelkers 1995;Hallén et al 1999;Hallén et al 2002b;Zhang et al 2004). Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al 2004) are glycosylated.…”

“…Both were cloned at the beginning of the 1990s and were termed Na + /taurocholate cotransporting polypeptide (NTCP; SLC10A1) (Hagenbuch et al 1990(Hagenbuch et al , 1991 and apical sodium-dependent bile acid transporter ASBT (SLC10A2), also called ileal Na + /bile acid cotransporter (ISBT, IBAT) (Wong et al 1994.…”

Age‐dependent glycosylation of the sodium taurocholate cotransporter polypeptide: From fetal to adult human livers

“…Structurally speaking, NTCP is a 55‐kDa N‐glycosylated multipass transmembrane protein composed of 349 amino acids that shares over 70% sequence similarity with rat Ntcp 14, 15. On the N‐terminal domain, two N‐linked glycosylation sites (Asn 5, Asn 11) facing the extracellular lumen have been characterized15, 16 and are important for NTCP localization on the plasma membrane 17.…”

“…On the N‐terminal domain, two N‐linked glycosylation sites (Asn 5, Asn 11) facing the extracellular lumen have been characterized15, 16 and are important for NTCP localization on the plasma membrane 17. Current knowledge of NTCP ontogenesis is mostly based on experiments conducted on rats and shows that i) compared to adult levels, Ntcp messenger RNA (mRNA) levels at birth are 35%‐76%; ii) in newborns, Ntcp protein expression reaches adult levels during the first postnatal week; iii) Ntcp expression levels and plasma membrane localization do not correlate with protein functional status; iv) NTCP activity is delayed approximately 4 weeks after birth 6, 18, 19, 20, 21.…”