Abstract
We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K(m) values of 28.7, 12.0, and 11.3 microm, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K(i) value of 0.24 mum. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(omega-sulfooxyethyl)pyrene, bromosulfophthalein, 2- and 4-sulfooxymethylpyrene, and alpha-naphthylsulfate. Among these inhibitors, 2- and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K(i) values of 4.3 and 5.5 microm, respectively, and they were also transported by SOAT-HEK293 cells.
Highlights
Sion cloning from rat liver [2] and is exclusively expressed at the sinusoidal membrane of hepatocytes [3, 4]
Sequence identity between NTCP and ASBT is quite low, both carriers transport conjugated bile acids with high affinity [7,8,9,10,11]. Due to their transport characteristics and expression pattern, NTCP and ASBT are important factors for the maintenance of the enterohepatic circulation of bile acids mediating the first step in the cellular uptake of bile acids through the membrane barriers in the liver (NTCP) and intestine (ASBT)
HEK293 cells, the ASBT open reading frame sequence was subcloned into the Flp-In pcDNA5/Flp recombinase target (FRT)/TO expression vector, and the FLAG epitope was inserted at the C-terminal end of ASBT by QuikChange site-directed mutagenesis as described above for sodium-dependent organic anion transporter (SOAT)
Summary
Materials and Chemicals—All of the chemicals, unless otherwise stated, were from Sigma. Cloning of Human SOAT cDNA—Using BLAST searches of the human genome with the cDNA sequences of the six coding exons of rat Soat (Slc10a6) (GenBankTM accession number AJ583503), we obtained matches with six genomic sequence fragments on human chromosome 4q21. The following oligonucleotide primers were designed, including SacII/XbaI restriction sites for PCR amplification: forward primer, 5Ј-atg acc gcg gat gag agc caa ttg ttc cag cag ctc-3Ј; reverse primer, 5Ј-cgt cta gac tat tcg cat gaa gtg atg tgg cca act g-3Ј. It has a relatively low expression in this organ (see below), human SOAT was initially cloned from the adrenal gland.
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